Whole-genome Phylogenetic Analysis Research Articles

Genomic epidemiology and transmission characteristics of mcr1-positive colistin-resistant Escherichia coli strains circulating at natural environment

MCR-positive Escherichia coli (MCRPEC) have been reported in humans worldwide. The high prevalence of mcr-1 poses clinical and environmental risks due to its diverse genetic mechanisms. Given the vital role of animals and the environment in the spread of antibiotic resistance, a “One Health” perspective should be taken when addressing antimicrobial resistance issues. This study conducted a prospective study in six farms (located in Jiaxing City, Zhejiang province, China) in 2019. MCRPEC strains were screened from samples of different sources. The molecular epidemiological surveys and transmission potential were investigated by whole-genome sequencing and phylogenetic analysis. MCRPEC were detected in different farms with various sources. Sequence type complex 10 was dominant and distributed widely in multiple sources. Core-genome multilocus sequence type (cgMLST) analysis indicated that clonal transmission could occur within and between farms. In addition, mcr-1 genes with different locations showed different transmission tendencies. The study indicated that interspecies and cross-regional transmission of MCRPEC could occur between different sectors in farms. Further surveillance and research of non-clinical MCRPEC strains are necessary to reduce the threat of MCRPEC.

Efflux Pumps and Different Genetic Contexts of tet(X4) Contribute to High Tigecycline Resistance in Escherichia fergusonii from Pigs.

Tigecycline is a last-resort antibiotic for the treatment of infections caused by multidrug-resistant bacteria. The emergence of plasmid-mediated tigecycline resistance genes is posing a serious threat to food safety and human health and has attracted worldwide attention. In this study, we characterized six tigecycline-resistant Escherichia fergusonii strains from porcine nasal swab samples collected from 50 swine farms in China. All the E. fergusonii isolates were highly resistant to tigecycline with minimal inhibitory concentration (MIC) values of 16-32 mg/L, and all contained the tet(X4) gene. In addition, 13-19 multiple resistance genes were identified in these isolates, revealed by whole-genome sequencing analysis. The tet(X4) gene was identified as being located in two different genetic structures, hp-abh-tet(X4)-ISCR2 in five isolates and hp-abh-tet(X4)-ΔISCR2-ISEc57-IS26 in one isolate. The role of efflux pumps in tigecycline resistance was evaluated by using inhibitor carbonyl cyanide 3-chlorophenylhydrazone (CCCP). The MIC values of tigecycline showed a 2- to 4-fold reduction in the presence of CCCP, indicating the involvement of active efflux pumps in tigecycline resistance in E. fergusonii. The tet(X4) gene was found to be transferable to Escherichia coli J53 by conjugation and resulted in the acquisition of tigcycline resistances in the transconjugants. Whole-genome multilocus sequence typing (wgMLST) and phylogenetic analysis showed a close relationship of five isolates originating from different pig farms, suggesting the transmission of tet(X4)-positive E. fergusonii between farms. In conclusion, our findings suggest that E. fergusonii strains in pigs are reservoirs of a transferable tet(X4) gene and provide insights into the tigecycline resistance mechanism as well as the diversity and complexity of the genetic context of tet(X4) in E. fergusonii.

1332. Population structure and genomic analysis of pediatric Streptococcus pyogenes clinical isolates in the United States, 2020 – 2022

Abstract Background Streptococcus pyogenes (GAS) is the major cause of bacterial pharyngitis in children. GAS can cause conditions ranging from asymptomatic pharyngeal carriage to invasive infection. We compared the genomic characteristics of GAS pharyngeal and invasive isolates from U.S. children. Methods We cultured GAS from throat swabs collected from children aged 3–18 years with acute GAS pharyngitis, diagnosed by rapid antigen or molecular test, and from a convenience sample of asymptomatic children during 1/2020 to 3/2022 in Chicago, IL; Atlanta, GA; Portland, OR; Phoenix, AZ. We collected invasive isolates in Chicago. Whole-genome sequencing (WGS) was performed on the Illumina MiSeq for species confirmation, isolate typing, and virulence and antimicrobial gene identification. Reference-based read alignment was used for whole genome phylogenetic analysis. Results Of 1144 collected throat swabs, 684 were from children with pharyngitis and 460 from asymptomatic children. 13 invasive GAS isolates were collected. GAS were cultured from 359 pharyngitis and 20 asymptomatic swabs. Of these, 371 GAS isolates could be recovered and underwent WGS. Thirty isolates (8%) were either low-quality sequences and/or identified as species other than S. pyogenes and were excluded. Whole-genome phylogenetic analysis of 341 GAS (215 pharyngitis, 13 asymptomatic, 13 invasive) identified 25 related clusters and 8 singletons (Figure 1). Virulence gene complements within given emm types were generally concordant with recently published CDC strain data. The most frequently identified resistance genes were tetM and ermB. 281 isolates (82%) carried no antibiotic resistance genes. Some clusters were significantly more represented at certain sites within apparent geographically defined clusters of nearly indistinguishable isolates (Figure 1): cluster 1 in Chicago, emm type 3; cluster 14 in Phoenix and Portland, emm 28, 12; and cluster 15 in Atlanta, emm 89. No major genomic factors were significantly associated with asymptomatic vs pharyngitis vs invasive presentation. Figure 1:Whole genome phylogenetic tree of pediatric GAS isolates Maximum likelihood phylogenetic analysis based on whole-genome alignment of 371 Group A Streptococcus isolates. Tip colors represent cluster assignments based on genetic similarity. Inner ring represents geographic location of GAS isolation. Outer ring represents patient presentation: asymptomatic colonization, symptomatic pharyngitis, or invasive infection. Conclusion Pharyngeal and invasive GAS in children within individual clonal complexes are co-included in closely related clusters, with evidence of close transmission within individual sites Disclosures All Authors: No reported disclosures.

Monkeypox in Montréal: Epidemiology, Phylogenomics, and Public Health Response to a Large North American Outbreak.

Monkeypox, a viral zoonotic disease, is causing a global outbreak outside of endemic areas. To characterize the outbreak of monkeypox in Montréal, the first large outbreak in North America. Epidemiologic and laboratory surveillance data and a phylogenomic analysis were used to describe and place the outbreak in a global context. Montréal, Canada. Probable or confirmed cases of monkeypox. Epidemiologic, clinical, and demographic data were aggregated. Whole-genome sequencing and phylogenetic analysis were performed for a set of outbreak sequences. The public health response and its evolution are described. Up to 18 October 2022, a total of 402 cases of monkeypox were reported mostly among men who have sex with men (MSM), most of which were suspected to be acquired through sexual contact. All monkeypox genomes nested within the B.1 lineage. Montréal Public Health worked closely with the affected communities to control the outbreak, becoming the first jurisdiction to offer 1 dose of the Modified Vaccinia Ankara-Bavarian Nordic vaccine as preexposure prophylaxis (PrEP) to those at risk in early June 2022. Two peaks of cases were seen in early June and July (43 and 44 cases per week, respectively) followed by a decline toward near resolution of the outbreak in October. Reasons for the biphasic peak are not fully elucidated but may represent the tempo of vaccination and/or several factors related to transmission dynamics and case ascertainment. Clinical data are self-reported. Limited divergence among sequences limited genomic epidemiologic conclusions. A large outbreak of monkeypox occurred in Montréal, primarily among MSM. Successful control of the outbreak rested on early and sustained engagement with the affected communities and rapid offer of PrEP vaccination to at-risk persons. None.

Plasmid-Coded Linezolid Resistance in Methicillin-Resistant Staphylococcus aureus from Food and Livestock in Germany

Resistance of methicillin-resistant Staphylococcus aureus (MRSA) from food and livestock to last resort antibiotics such as linezolid is highly concerning, since treatment options for infections in humans might be diminished. Known mechanisms of linezolid resistance include point mutations in the 23S rRNA gene and in the ribosomal proteins L3, L4 and L22 as well as an acquisition of the cfr, optrA or poxtA gene. The objective of our study was to characterize antimicrobial resistance (AMR) determinants and phylogenetic relationships among linezolid-resistant (LR-) MRSA from food and livestock. In total, from more than 4000 incoming isolates in the years 2012 to 2021, only two strains from 2015 originating from pig samples exhibited linezolid resistance in the antimicrobial susceptibility testing with MICs of ≥8 mg/L. These LR-MRSA were characterized in detail by whole-genome sequencing and phylogenetic analyses using cgMLST. The LR-MRSA strains showed resistances to ten and eight different antibiotics, respectively. Both strains harbored plasmid-coded cfr genes mediating the linezolid resistance. The cfr genes showed identical sequences in both strains. In addition to the cfr gene, genes for phenicol and clindamycin resistance were detected on the respective plasmids, opening the possibility for a co-selection. The LR-MRSA differed distantly in the phylogenetic analyses and also to other MRSA from pig samples in the year 2015. In conclusion, the occurrence of LR-MRSA in food and livestock seems to be very rare in Germany. However, carriage of plasmids with linezolid resistance determinants could lead to further linezolid-resistant strains by horizontal gene transfer.

Lacrimispora defluvii PI-S10-B5AT sp. nov., an Obligate Anaerobe, Isolated from an Industrial Waste and Reclassification of Hungatella xylanolytica as Lacrimispora xylanolytica and Clostridium indicum as Lacrimispora indica Comb. nov.

A bacterial strain was isolated from the waste slurry of an industrial effluent treatment plant near Patancheru, Hyderabad, India, and designated as PI-S10-B5AT. It was an obligately anaerobic, spore-forming, rod-shaped, motile bacterium that stained Gram-positive. The strain revealed high 16S rRNA gene sequence identity with Hungatella xylanolytica DSM 3808T (99.4%) followed by members of the genus Lacrimispora (98.8-93.3%). However, the average nucleotide identity (ANI) and digital DNA-DNA hybridization of genome sequence exhibited similarity in the range of 94.3-68.7% and 57.4-18.8%, respectively, with all closely related strains. A multi-gene phylogenetic analysis of strain PI-S10-B5AT was performed to investigate the taxonomic affiliation, which revealed formation of a coherent cluster with the members of the genus Lacrimispora. The DNA G + C content was 41.8mol%. Major polar lipids were glyco- and phospholipids. The fatty acids analysis showed C16:0 to be the major fatty acid. The predominant respiratory quinone was menaquinone-7 (MK-7). Based on phenotypic, chemotaxonomic, and whole-genome phylogenetic analysis, strain PI-S10-B5AT is assigned as a novel species of the genus Lacrimispora, for which the name Lacrimispora defluvii is proposed. The type strain of the novel species is PI-S10-B5AT (= MTCC 12280T; = DSM 24980T) isolated from waste slurry of effluent treatment plant. The genomic analysis of type strains of C. indicum PI-S10-A1BT and H. xylanolytica DSM 3808T showed ANI and AAI values consistent with members of the genus Lacrimispora. Therefore, these strains are ascertained to the genus Lacrimispora and reclassified as Lacrimispora indica and Lacrimispora xylanolytica comb. nov.

In silico, in vitro and in vivo characterization of host-associated Latilactobacillus curvatus strains for potential probiotic applications in farmed Atlantic salmon (Salmo salar)

Salmon aquaculture is the fastest growing animal protein production system in the world; however, intensive farming leads to poor weight gain, stress, and disease outbreaks. Probiotics offer the potential to enhance growth performance and feed efficiency in Atlantic salmon, as well as immunostimulate fish against common pathogens, benefitting farmers and consumers with more efficient production. Here, we isolated and identified 900 native microbial isolates including 18 Lactobacilli from the farmed salmon intestines. Based on whole-genome sequencing and phylogenetic analysis, the Lactobacillus candidates belonged to Latilactobacillus curvatus (L. curvatus) species and formed two distinct phylogenetic groups. Using bioinformatics and in vitro analyses, we selected two candidates L. curvatus ATCC PTA-127116 and L. curvatus ATCC PTA-127117, which showed desirable safety and probiotic properties. The two L. curvatus candidates were evaluated for safety and efficacy (higher final weight) in Atlantic salmon alongside spore-forming Bacilli isolated from salmon, poultry, and swine. All the tested candidates were safe to salmon with no adverse effects. While we did not see efficacy in any Bacillus supplemented groups, compared to untreated group, the group administered with the two L. curvatus strains consortium in feed for seven weeks in freshwater showed indicators of improvement in final body weight by 4.2%. Similarly, the two L. curvatus candidates were also evaluated for safety and efficacy in Atlantic salmon in saltwater; the group administered with the two L. curvatus strains consortium in feed for 11 weeks showed indicators of improvement in final body weight by 4.7%. Comprehensive metabolomics analyses in the presence of different prebiotics and/or additives identified galactooligosaccharide as a potential prebiotic to enhance the efficacy of two L. curvatus candidates. All together, these data provide comprehensive genomic, phenotypic and metabolomic evidence of safety and desirable probiotic properties as well as indicators of in vivo efficacy of two novel endogenous L. curvatus candidates for potential probiotic applications in Atlantic salmon. The in vivo findings need to be confirmed in larger performance studies, including field trials.

Whole-genome analysis of human papillomavirus 67 isolated from Japanese women with cervical lesions

BackgroundHuman papillomavirus (HPV) type 67 is phylogenetically classified into Alphapapillomavirus species 9 (alpha-9) together with other carcinogenic types (HPV16, 31, 33, 35, 52 and 58), but is the only alpha-9 type defined as possibly carcinogenic. This study aimed to determine whole-genome sequences of HPV67 isolated from Japanese women with cervical cancer or cervical intraepithelial neoplasia (CIN) to better understand the genetic basis of the oncogenic potential of HPV67.MethodsTotal cellular DNA isolated from cervical exfoliated cells that were single positive for HPV67 (invasive cervical cancer, n = 2; CIN3, n = 6; CIN 2, n = 1; CIN1, n = 2; the normal cervix, n = 1) was subjected to PCR to amplify HPV67 DNA, followed by next generation sequencing to determine the complete viral genome sequences. Variant lineages/sublineages were assigned through viral whole-genome phylogenetic analysis. The transcriptional activity of the virus early promoter was assessed by luciferase reporter assays in cervical cancer cell lines.ResultsPhylogenetic analyses of HPV67 genomes from Japan (n = 12) revealed that 11 belonged to lineage A (sublineage A1, n = 9; sublineage A2, n = 2) and one belonged to lineage B. All cancer cases contained sublineage A1 variants, and one of these contained an in-frame deletion in the E2 gene. The long control region of the HPV67 genome did not induce transcription from the virus early promoter in HeLa cells, but showed weak transcriptional activity in CaSki cells.ConclusionsThe single detection of HPV67 in cervical cancer and precancer specimens strongly suggests the carcinogenic potential of this rare genotype. The phylogenetic analysis indicates a predominance of lineage A variants among HPV67 infections in Japan. Since only 23 complete genome sequences of HPV67 had been obtained until now, the newly determined genome sequences in this study are expected to contribute to further functional and evolutionary studies on the genetic diversity of HPV67.

Reference quality genome sequence of Indian pomegranate cv. 'Bhagawa' (Punica granatum L.).

Pomegranate is an important fruit crop for ensuring livelihood and nutrition security in fragile semi-arid regions of the globe having limited irrigation resources. This is a high-value, nutritionally rich, and export-oriented agri-commodity that ensures high returns on investment to growers across the world. Although it is a valuable fruit crop, it has received only a limited genomics research outcome. To fast-track the pomegranate improvement program, de novo whole-genome sequencing of the main Indian cultivar ‘Bhagawa’ was initiated by the Indian Council of Agricultural Research–National Research Center on Pomegranate (ICAR–NRCP). We have demonstrated that a combination of commercially available technologies from Illumina, PacBio, 10X Genomics, and BioNano Genomics could be used efficiently for sequencing and reference-grade de novo assembly of the pomegranate genome. The research led to a final reference-quality genome assembly for ‘Bhagawa’ of 346.08 Mb in 342 scaffolds and an average N50 of 16.12 Mb and N90 of 1088.62 Kb. This assembly covered more than 98% of the estimated pomegranate genome size, 352.54 Mb. The LTR assembly index (LAI) value of 10 and 93.68% Benchmarking Universal Single-Copy Orthologs (BUSCO) completeness score over the 1,440 ortholog genes of the completed pomegranate genome indicates the quality of the assembled pomegranate genome. Furthermore, 29,435 gene models were discovered with a mean transcript length of 2,954 bp and a mean coding sequence length 1,090 bp. Four transcript data samples of pomegranate tissues were mapped over the assembled ‘Bhagawa’ genome up to 95% significant matches, indicating the high quality of the assembled genome. We have compared the ‘Bhagawa’ genome with the genomes of the pomegranate cultivars ‘Dabenzi’ and ‘Taishanhong.’ We have also performed whole-genome phylogenetic analysis using Computational Analysis of Gene Family Evolution (CAFE) and found that Eucalyptus grandis and pomegranate diverged 64 (60–70) million years ago. About 1,573 protein-coding resistance genes identified in the ‘Bhagawa’ genome were classified into 32 domains. In all, 314 copies of miRNA belonging to 26 different families were identified in the ‘Bhagawa’ genome. The reference-quality genome assembly of ‘Bhagawa’ is certainly a significant genomic resource for accelerated pomegranate improvement.

Genome-based reclassification of Anoxybacillus kamchatkensis Kevbrin et al. 2005 as a later heterotypic synonym of Anoxybacillus ayderensis Dulger et al. 2004.

In this study, we aimed to clarify the taxonomic positions of Anoxybacillus kamchatkensis DSM 14988T and Anoxybacillus ayderensis AB04T using whole-genome phylogenetic analysis, biochemical and chemotaxonomic characteristics. In phylogenetic trees drawn using whole-genome sequences and 16S rRNA gene sequences, A. kamchatkensis DSM 14988T and A. ayderensis AB04T clade together and showed high sequence similarity (99.6%) based on 16S rRNA gene. The average amino acid identity, average nucleotide identity and digital DNA-DNA hybridization values between A. kamchatkensis DSM 14988T and A. ayderensis AB04T were found to be greater than the threshold values for species demarcation. Most phenotypic and chemotaxonomic features between both species were almost identical except for a few exceptions. The present results show that A. kamchatkensis DSM 14988T is a later heterotypic synonym of A. ayderensis AB04T.

Late-Onset Sepsis in a Premature Infant Mediated by Breast Milk: Mother-to-Infant Transmission of Group B Streptococcus Detected by Whole-Genome Sequencing.

BackgroundLate-onset group B Streptococcus (LOGBS) sepsis is a cause of infection and death in infants. Infected breast milk has been considered a source of neonatal GBS infection and invasive infection. However, mother-to-infant transmission of GBS detected by the high-resolution diagnostic method is rarely reported.MethodsThis study describes a low-weight premature infant who developed late-onset GBS septicemia 21 days after birth. GBS strains isolated from the mother’s cervical secretion, the mother’s milk, and the baby’s blood were cultured to identify the source of GBS infection. We further confirmed the GBS isolates through matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS). Finally, we performed whole-genome sequencing (WGS) and phylogenetic analyses on the GBS strains recovered.ResultsGBS isolates were cultured from the bloodstream of the premature infant and the mother’s milk, respectively. Subsequently, WGS and phylogenetic analyses on three GBS isolates demonstrated that the GBS strain from the infant’s bloodstream was 100% homologous to that from the mother’s breast milk, which had some different gene fragments from the GBS strain from the mother’s cervical secretion. It provided evidence that this infant’s late-onset GBS septicemia originated from his mother’s breast milk instead of the vertical mother-to-infant transmission.ConclusionThrough WGS and phylogenetic analysis of the GBS strains, we proved in this study that the late-onset GBS sepsis in a premature infant was derived from his mother’s breast milk. It indicated that WGS diagnosis is an effective tool for infection tracing. Furthermore, this report provides direction for preventing late-onset GBS infection.

Genetic and epidemic characteristics of porcine parvovirus 7 in the Fujian and Guangdong regions of southern China.

Porcine parvovirus (PPV) is the primary cause of reproductive disorders in pigs. The porcine parvovirus 7 (PPV7) subtype was first identified in the United States in 2016. In this study, PPV7 was detected in different porcine samples, including serum, feces, saliva, and milk, from 69 pig farms in the Fujian and Guangdong regions of South China, and its coinfection with porcine circovirus 2 (PCV2), porcine circovirus 3 (PCV3), and porcine reproductive and respiratory syndrome virus (PRRSV) was determined. Whole-genome sequencing, phylogenetic analysis, and recombination analysis were performed on seven isolates, with each selected isolate originating from a different farm. There was a high rate of PPV7 positivity in blood, stool, and saliva but PPV7 DNA was absent from breast milk. The findings also showed that PPV7-positive samples had a high rate of coinfection with PCV2, PCV3, and PRRSV. Real-time PCR was used to determine the viral copy numbers of PCV2, PCV3, PRRSV, and PPV7 in serum samples and to assess whether PPV7 affected PCV2, PCV3, and PRRSV viral loads. Phylogenetic analysis showed that PPV7e and PPV7f were the most prevalent and widespread subtypes in the Fujian and Guangdong regions, respectively. While the PPV7a, PPV7b, PPV7c, and PPV7f subtypes were most prevalent in Fujian Province, PPV7a-e subtypes were prevalent in Guangdong, indicating that PPV7 has rich genetic diversity in these regions. A putative recombinant strain, 21FJ09, was identified using SimPlot and the Recombination Detection Program 4 software.

Isolation and characterization of an orthoreovirus from Indonesian fruit bats

Nelson Bay orthoreovirus (NBV) is an emerging bat-borne virus and causes respiratory tract infections in humans sporadically. Over the last two decades, several strains genetically related to NBV were isolated from humans and various bat species, predominantly in Southeast Asia (SEA), suggesting a high prevalence of the NBV species in this region. In this study, an orthoreovirus (ORV) belonging to the NBV species was isolated from Indonesian fruit bats' feces, tentatively named Paguyaman orthoreovirus (PgORV). Serological studies revealed that 81.2% (108/133) of Indonesian fruit bats sera had neutralizing antibodies against PgORV. Whole-genome sequencing and phylogenetic analysis of PgORV suggested the occurrence of past reassortments with other NBV strains isolated in SEA, indicating the dispersal and circulation of NBV species among bats in this region. Intranasal PgORV inoculation of laboratory mice caused severe pneumonia. Our study characterized PgORV's unique genetic background and highlighted the potential risk of PgORV-related diseases in Indonesia.

Influence of genomic structural variations and nutritional conditions on the emergence of quorum sensing-dependent gene regulation defects in Burkholderia glumae.

Bacteria often change their genetic and physiological traits to survive in harsh environments. To determine whether, in various strains of Burkholderia glumae, genomic diversity is associated with the ability to adapt to ever-changing environments, whole genomes of 44 isolates from different hosts and regions were analyzed. Whole-genome phylogenetic analysis of the 44 isolates revealed six clusters and two divisions. While all isolates possessed chromosomes 1 and 2, strains BGR80S and BGR81S had one chromosome resulting from the merging of the two chromosomes. Upon comparison of genomic structures to the prototype BGR1, inversions, deletions, and rearrangements were found within or between chromosomes 1 and/or 2 in the other isolates. When three isolates—BGR80S, BGR15S, and BGR21S, representing clusters III, IV, and VI, respectively—were grown in Luria-Bertani medium, spontaneous null mutations were identified in qsmR encoding a quorum-sensing master regulator. Six days after subculture, qsmR mutants were found at detectable frequencies in BGR15S and BGR21S, and reached approximately 40% at 8 days after subculture. However, the qsmR mutants appeared 2 days after subculture in BGR80S and dominated the population, reaching almost 80%. No qsmR mutant was detected at detectable frequency in BGR1 or BGR13S. The spontaneous qsmR mutants outcompeted their parental strains in the co-culture. Daily addition of glucose or casamino acids to the batch cultures of BGR80S delayed emergence of qsmR mutants and significantly reduced their incidence. These results indicate that spontaneous qsmR mutations are correlated with genomic structures and nutritional conditions.

Genome-based reclassification of Anoxybacillus karvacharensis Panosyan et al. as a later heterotypic synonym of Anoxybacillus kestanbolensis Dulger et al. 2004; A. flavithermus subsp. yunnanensis CCTCC AB2010187T Dai et al. 2011 as a later heterotypic synonym of A. tengchongensis DSM 23211T Zhang et al. 2011.

In the present study, we attempted to clarify the taxonomic positions of Anoxybacillus karvacharensis K1T, Anoxybacillus kestanbolensis NCIMB 13971T, Anoxybacillus flavithermus subsp. yunnanensis CCTCC AB2010187T, and Anoxybacillustengchongensis DSM 23211T using whole-genome phylogenetic analysis. The genome sequence of A. kestanbolensis NCIMB13971T was not available in any database, so it was sequenced in this study. The 16S rRNA gene sequence obtained from the genome of A. kestanbolensis NCIMB13971T had 99.93% similarity with A. karvacharensis K1T. The average nucleotide identity (ANI), average amino acid identity (AAI), and digital DNA-DNA hybridization (DDH) values between A. karvacharensis K1T and A. kestanbolensis NCIMB13971T and between A. flavithermus subsp. yunnanensis CCTCCAB 2010187T and A. tengchongensis DSM 23211T were greater than the threshold values for species demarcation. The present results indicate that A. karvacharensis K1T is a later heterotypic synonym of A. kestanbolensis NCIMB13971T; A. flavithermus subsp. yunnanensis CCTCCAB 2010187T is a later heterotypic synonym of A. tengchongensis DSM 23211T.

Explosive outbreak of SARS-CoV-2 Omicron variant is associated with vertical transmission in high-rise residential buildings in Hong Kong

The phenomenon of vertical transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in high-rise residential buildings (HRRBs) is unique in our densely populated cosmopolitan city. The compulsory testing of a whole building under the scheme of restriction-testing declaration (RTD) during the fourth wave (non-Omicron variant) and fifth wave (mostly Omicron variant) of COVID-19 outbreak in Hong Kong allowed us to study the prevalence of this phenomenon, which may represent a form of airborne transmission. From 23 January 2021 to 24 March 2022, 25,450 (5.8%) of 436,397 residents from 223 (63.0%) of 354 HRRBs under RTD were test-positive for SARS-CoV-2. Using the clustering of cases among vertically aligned flats with shared drainage stack and lightwell as a surrogate marker of vertical transmission, the number of vertically aligned flats with positive COVID-19 cases was significantly higher in the fifth wave compared with the fourth wave (14.2%, 6471/45,531 vs 0.24%, 3/1272; p < 0.001; or 2212 vs 1 per-million-flats; p < 0.001). Excluding 22,801 residents from 38 HRRBs who were tested negative outside the 12-week periods selected in fourth and fifth waves, the positive rate among residents was significantly higher among residents during the fifth wave than the fourth wave (6.5%, 25,434/389,700 vs 0.07%, 16/23,896; p < 0.001). Within the flats with COVID-19 cases, the proportion of vertically aligned flats was also significantly higher in the fifth wave than in the fourth wave (95.6%, 6471/6766 vs 30.0%, 3/10, p < 0.001). The proportion of HRRBs with COVID-19 cases was significantly higher during the corresponding 12-week period chosen for comparison (78.2%, 219/280 vs 11.1%, 4/36; p < 0.001). Whole-genome phylogenetic analysis of 332 viral genomes showed that Omicron BA.2 was the predominant strain, supporting the high transmissibility of BA.2 by airborne excreta-aerosol route in HRRBs of Hong Kong.

Epidemiological and genetic characteristics of clinical carbapenem-resistant Acinetobacter baumannii strains collected countrywide from hospital intensive care units (ICUs) in China

ABSTRACT Acinetobacter baumannii is one of the key Gram-negative pathogens that can cause serious nosocomial infections. In China, a large proportion of clinical A. baumannii strains are multidrug resistant, among which strains resistant to carbapenems are particularly worrisome, as infections caused by such strains may limit the choice of existing antibiotics. We conducted a nationwide and genome-based surveillance on the prevalence and antibiotic susceptibility profile of carbapenem-resistant A. baumannii (CRAB) strains collected from intensive care units (ICUs) in hospitals in different provinces and investigated the routes of transmission and mechanism of resistance by whole-genome sequencing and phylogenetic analysis. We found that CRAB strains were prevalent in 71.4% (55/77) of the ICUs surveyed. Clonal spread of CRAB was found in 37.6% (29/77) of ICUs and a total of 22 different clones were identified. Most clones were transmissible within one ICU, but up to six clones could be detected in at least three hospitals. In addition, carbapenem-hydrolysing class D β-lactamases (CHDL) were found to be mainly responsible for carbapenem-resistance in A. baumannii and the ST2 global-clone is the predominant type of CRAB in China. Importantly, we found that CRAB isolates currently exhibited an extremely low rate of resistance to colistin (0.4%) and tigecycline (2.5%), but a high rate of resistance to ceftazidime–avibactam (70.2%). Findings in this work shall facilitate development of appropriate antimicrobial regimens for treatment of CRAB infections. Further surveillance and research on the evolutionary and epidemiological features of clinical CRAB strains are necessary.

Influenza A in Wild Boars: Viral Circulation in the Emilia-Romagna Region (Northern Italy) between 2017 and 2022.

Simple SummaryWild boars and feral pigs are underinvestigated hosts for influenza A viruses (IAVs). This study confirmed and evaluated viral circulation in the Emilia-Romagna wild boar population between 2017 and 2022. Samples were collected at post mortems and screened for IAVs; 0.37% of the tested animals provided positive results. Positive samples were subtyped, isolated, and genotyped via full-genome sequencing. The results highlight the co-circulation of the same viral genotypes in overlapping years in both pigs and wild boars in the same geographical area. Considering the role of domestic and wild Sus scrofa species in the IAVs’ ecology, surveillance against these viruses in the wild boar population needs to be implemented.A systematic surveillance against influenza A viruses (IAVs) in the Suidae population is essential, considering their role as IAV mixing vessels. However, the viral circulation in wild Sus scrofa species is poorly investigated in comparison to the knowledge of IAV infection dynamics in domestic pigs. This study investigated the circulation and the genetic diversity of wild boars’ IAVs detected in the Emilia-Romagna region (2017–2022). A total of 4605 lung samples were screened via an M gene real-time RT-PCR for SwIAV; positive samples were subtyped by multiplex RT-PCR, and viral isolation was attempted. Isolated strains (3 out of the 17 positives) were fully sequenced to evaluate viral genotypic diversity. H1N1 was the most frequently detected subtype, with identification of H1pdm09N1 and H1avN1. Whole-genome phylogenetic analysis revealed SwIAVs belonging to different genotypes, with different genetic combinations, and highlighted the simultaneous circulation of the same genotypes in both pigs and wild boars, supporting the hypothesis of SwIAV spillover events at the wildlife–livestock interface. This study represents an update on the wild boar SwIAV Italian situation, and the strains’ complete genome analysis showed an evolving and interesting situation that deserves further investigation.

Detection of NDM-1-Positive Aeromonas caviae from Bacteremia by Using Whole-Genome Sequencing.

PurposeNosocomial infections caused by New Delhi metallo-β-lactamase (NDM)-producing bacteria are prevalent worldwide. However, such diseases caused by NDM-producing Aeromonas caviae had never been reported. Our study aimed to elucidate the genomic characteristics of NDM-1-producing A. caviae isolated from hospital patients.MethodsBacterial genomic features and possible origins were assessed by whole-genome sequencing (WGS) and phylogenetic analysis. Subsequent investigations include antimicrobial susceptibility testing and multilocus sequence typing (MLST).ResultsWe identified here two NDM-1-producing A. caviae isolates from bacteremia. Susceptibility testing showed that two isolates were multi-drug resistant and shared a similar resistance profile and were only sensitive to amikacin and trimethoprim/sulfamethoxazole. Both A. caviae isolates carry the carbapenem resistance gene blaNDM-1 and also have antibiotic resistance genes such as β-lactams, AmpC enzymes, macrolides, aminoglycosides, and quinolones. S1-PFGE and Southern blot analysis were negative. Whole-genome sequencing and comparative analysis revealed that these two isolates shared a close relationship.ConclusionTo the best of our knowledge, this work describes the first detection of non-plasmid encoded blaNDM-1 in A. caviae. The A. caviae isolated in this study has a broad drug resistance spectrum. Phenotypic and molecular analysis indicated the two isolates belong to the same clone. Routine genomic surveillance of this species is now necessary to effectively curb the further dissemination of carbapenem-resistant bacteria in the region.

Detection of SARS-CoV-2 Reinfections by Rapid Inexpensive Methods

New SARS-CoV-2 infections are difficult to beverified, whether they are reinfections or persistent infections. The most prominent factors used for differentiating reinfections from persistent infections are whole-genome sequencing and phylogenetic analyses that require time and funds, which may not be feasible in most developing countries. This study explores reinfections with COVID-19 that harbors D614G and N501Y mutations by rapid inexpensive methods. It exploits the previously developed rapid economic methods that identified both D614G and N501Y mutations in clinical samples using real-time reverse transcriptase polymerase chain reaction (rRT-PCR) probes and conventional PCR specific primers. In the present study, an immunocompetent patient has been found with a SARS-CoV-2 N501Y reinfection without comorbidities. According to the obtained results, this study suggests that the initial infection was due to a variant that contained only D614G mutation whereas the reinfection was potentially a result of alpha variant contained three mutations confirmed by DNA sequencing, including D614G, N501Y, and A570D mutations. These techniques will support rapid detection of SARS-CoV-2 reinfections through the identification of common spike mutations in the developing countries where sequencing tools are unavailable. Furthermore, seven cases of reinfections were also confirmed by these methods. These rapid methods can also be applied to large samples of reinfections that may increase our understanding epidemiology of the pandemic.