Phylogenetic Typing Research Articles

Characterization of non-O157 enterohemorrhagic Escherichia coli isolated from different sources in Egypt

BackgroundEnterohemorrhagic Escherichia coli (EHEC) O157 is implicated in serious food and water-borne diseases as hemorrhagic colitis (HC), and the potentially fatal hemolytic uremic syndrome (HUS). However, new players of non-O157 EHEC have been implicated in serious infections worldwide. This work aims at analyzing serotype and genotypic-based virulence profile of EHEC local isolates.MethodsA total of 335 samples were collected from different sources in Egypt. E. coli was isolated and subjected to serotyping. Non-O157 EHEC isolates were tested for virulence genes using PCR, phenotypic examination, phylogenetic typing, and molecular investigation by ERIC typing and MLST to disclose genetic relatedness of isolates. A heat map was used to identify potential associations between the origin of the isolates, their phenotypic and genotypic characteristics.ResultsA total of 105 out of 335 isolates were identified as E. coli. Surprisingly, 49.5% of these isolates were EHEC, where O111, O91, O26 and O55 were the most prevalent serotypes including 38.46% from stool, 21.15% urine, 23.1% cheese, 9.62% meat products, 3.85% from both yogurt and sewage water. Screening 15 different virulence genes revealed that sheA, stx2 and eae were the most prevalent with abundance rates of 85%, 75% and 36%, respectively. Fifteen profiles of virulence gene association were identified, where the most abundant one was stx2/sheA (19%) followed by stx2/stx2g/sheA/eae (11.5%). Both stx2/sheA/eae and stx2/stx2g/sheA were equally distributed in 9.6% of total isolates. Phylogenetic typing revealed that pathogenic phylogroups B2 and D were detected among clinical isolates only. Forty-six different patterns were detected by ERIC genotyping. MLST resolved three sequence types of ST70, ST120 and ST394. The heat map showed that 21 isolates were of 70% similarity, 9 groups were of 100% clonality.ConclusionsThe prevalence of non-O157 EHEC pathotype was marginally higher among the food isolates compared to the clinical ones. The endemic ST120 was detected in cheese, necessitating crucial measures to prevent the spread of this clone. Clinical EHEC isolates exhibited a higher score, and combination of virulence genes compared to food and sewage water isolates, thereby posing a significant public health concern.

Quinolone and Tetracycline-Resistant Biofilm-Forming Escherichia coli Isolates from Slovak Broiler Chicken Farms and Chicken Meat

Escherichia coli isolates from intensive poultry production are associated with antimicrobial resistance and worldwide health problems. The aim of the study was to detect and evaluate the phenotypic and genotypic antimicrobial resistance, biofilm formation, phylogenetic typing, and virulence factors in E. coli isolates from the rectal swabs of chickens from two farms and swabs of chicken meat purchased from Slovakian food markets. Interpretative readings of minimal inhibitory concentration (MIC) revealed dominant resistance to ampicillin (>50%) in both groups. We also detected higher resistance to ciprofloxacin (45%), tetracycline, ampicillin + sulbactam, and trimethoprim + sulfonamide (each >30%). Here, 28.57% of the strains studied were multidrug-resistant (MDR). The formation of weak biofilms was confirmed in 8.8% of E. coli, while one of the strains obtained from chicken cloacal swabs was classified as a strong biofilm producer. The most frequently confirmed phylogenetic groups in E. coli were B1 and A1 in all groups. PCR detection revealed the presence of genes encoding tetracycline resistance (tetAB) and plasmid-mediated quinolone resistance (qnrABS), and Int1 (52.9%), Tn3 (76.5%), kpsMT II (8.8%), fimA (97.1%), cvaC (38.2%), and iutA (76.5%) genes in the strains studied. Our results demonstrate that chickens and chicken meat were the source of antibiotic-resistant, biofilm-forming, and virulent E. coli, representing a potential risk from the point of view of the One Health concept.

Comparison of CRISPR typing and conventional molecular methods for distinguishing Laribacter hongkongensis isolates from fish, frogs and humans

High-resolution and efficient typing for Laribacter hongkongensis (L. hongkongensis) is essential for epidemiological investigation of such emerging foodborne pathogens. Clustered regularly interspaced short palindromic repeats (CRISPR) typing is an innovative molecular method that shows great promise for L. hongkongensis typing. Here, we explored the CRISPR typing method by combining CRISPR1 and CRISPR2 loci to characterize a collection of 109 L. hongkongensis isolates from humans and animals and compared it to current molecular methods such as pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The results showed that all three methods have high discriminatory power (diversity index was 0.9902 for PFGE, 0.9663 for CRISPR and 0.9562 for MLST); strong congruence was observed between them (Rand index was 0.969 between CRISPR and PFGE, 0.953 between CRISPR and MLST, 0.958 between PFGE and MLST). CRISPR typing could well distinguish the isolates in the same STs or PFGE profiles, and the genetic information contained by the CRISPR array is useful for deep phylogenetic typing. We demonstrate that rapid CRISPR typing is a practical genetic fingerprinting tool with high resolution, comparable ease of use and lower cost, ability to track the source of various groups of L. hongkongensis strains and indication of genetic characteristics.

The phylogroups and antibiotic susceptibilities of Escherichia coli isolates from the feces of Anatolian Ground Squirrels (Spermophilus xanthoprymnus)

The current study was conducted to determine the phylogroups and antibiotic susceptibilities of Escherichia coli isolates recovered from fecal samples of Anatolian Ground Squirrels (Spermophilus xanthoprymnus) and to examine the relationship between them. Eighty-two E. coli isolates obtained from 150 fecal samples were investigated. The quadruplex polymerase chain reaction (PCR), phylogroup C-, and E-specific mPCR were subjected to phylogenetic typing of the isolates. The susceptibilities to fifteen antibiotics of the isolates were detected by the disk diffusion method. In the result of phylogenetic typing, phylogroup B2 was most predominant (58.6 %), followed by B1 (25.6 %), E (8.5 %), C (4.9 %), and D (2.4 %). The phylogroup A, F, and Escherichia clades were not detected. The antibiotic susceptibility test revealed that 59.8 % (49/82) and 19.5 % (16/82) of E. coli isolates were resistant to at least one antibiotic and multidrug-resistant (MDR), respectively. Twenty-six (31.7 %), 19 (23.2 %), 11 (13.4 %), and 10 (12.2 %) of the isolates were found to be resistant to gentamicin, tetracycline, amoxicillin-clavulanic acid, and cefoxitin. Of the 49 E. coli isolates that were found to be resistant to any antibiotic analyzed, 30, 13, 4, and 2 were located in phylogroup B2, B1, E, and D, respectively. MDR isolates were mostly located in both phylogroup B1 (31.3 %) and B2 (31.3 %). In conclusion, data from the current study suggest that the isolates may potentially have pathogenic properties, since the majority (69.5 %) of E. coli isolates from fecal samples of Spermophilus xanthoprymnus were located in the pathogenic phylogroup and resistance to various antibiotics was detected.

A clade of Streptococcus pneumoniae clonal complex 320 with increased tolerance to β-lactam antibiotics in a Chinese metropolitan city

ObjectivesWe characterized the population structure and features of clinical Streptococcus pneumoniae isolates associated with invasive pneumococcal disease (IPD) from 2009 to 2017 in a Chinese metropolitan city using a whole-genome sequencing approach. MethodsSeventy-nine pneumococcal strains, including 60 serogroup-19 strains from children enduring IPD from a paediatric hospital in Shenzhen, were subjected to whole-genome sequencing. Population structure was characterized through phylogenetic analysis, sequence typing, serotyping, virulence factor, and antimicrobial drug resistance (AMR) gene profiling, combining the publicly available related WGS data. Clinical demography and antibiotic susceptibility profiles were compared among different populations to emphasize the higher-risk populations. Genetic regions associated with AMR gene mobilization were identified through comparative genomics. ResultsThese IPD strains mainly belonged to clonal complex 320 (CC320) and were composed of serotypes 19A and 19F. In addition to sporadic possible importation-related isolates (ST320), we identified an independent clade, CC320_SZpop (ST271), that predominantly circulated in Shenzhen and possibly expanded its range. Clinical features and antibiotic susceptibility analysis revealed that CC320_SZpop might manifest much higher pathogenicity and tolerance to β-lactams. Specific virulence factors in Shenzhen isolates of CC320_SZpop were identified. Furthermore, an ca. 40 kb hotspot genomic region enduring frequent recombination was identified, possibly associated with the divergence of S. pneumoniae strains. ConclusionA novel pneumococcal clade, CC320_SZpop, circulating in Shenzhen and other regions in China, possibly under expansion, was found and deserves more study and surveillance. Our study also emphasizes the importance of continuous genomic surveillance of clinical S. pneumoniae isolates, especially IPD isolates.

Molecular identification and characterization and phylogenetic study in Escherichia coli in Baghdad province

The study involved a total of 112 Escherichia coli isolates that were obtained from diverse origins. It took place within the jurisdiction of Baghdad Province. The isolates were classified into distinct phylogenetic groups using Quadruplex Clermont's innovative methodology. Phylogenetic typing was determined using the multiplex PCR technique. The findings of the present investigation reveal that the phylogenetic group B2 exhibited the highest prevalence rate (34.8%), followed by phylogenetic groups C (21.4%), D (16.9%), and A (10.7%). The remaining groups, namely B1 (5.3%), E (4.4%), F (2.6%), and unknown (3.5%), exhibited comparatively lower prevalence rates. Furthermore, the present study has identified a significant correlation between cephalosporins and phylogenetic groupings of E. coli. The cephalosporin groups were found to be associated with a significant proportion of antibiotic resistance patterns, whereas the carbapenem groups exhibited the lowest association. The biofilm formation was detected in 78.5% (88/112) of the isolates, and 95.5% (107/112) of the strains exhibited multiresistance. The group B2 exhibited the highest prevalence of antibiotic resistance and biofilm formation, with a percentage of 33.6% (36/107) and 35% (31/88).

Genomic analysis of acid tolerance genes and deciphering the function of ydaG gene in mitigating acid tolerance in Priestia megaterium.

Adverse environmental conditions, such as acid stress, induce bacteria to employ several strategies to overcome these stressors. These strategies include forming biofilms and activating specific molecular pathways, such as the general stress response (GSR). The genome of Priestia megaterium strain G18 was sequenced using the Illumina NextSeq 500 system, resulting in a de novo assembly of 80 scaffolds. The scaffolded genome comprises 5,367,956 bp with a GC content of 37.89%, and was compared to related strains using the MiGA web server, revealing high similarity to P. megaterium NBRC 15308 and P. aryabhattai B8W22 with ANI scores of 95.4%. Phylogenetic and ribosomal multilocus sequence typing (rMLST) analyses, based on the 16S rRNA and ribosomal protein-encoding alleles, confirmed close relationships within the P. megaterium species. Functional annotation identified 5,484 protein-coding genes, with 72.31% classified into 22 COG categories, highlighting roles in amino acid transport, transcription, carbohydrate metabolism, and ribosomal structure. An in-depth genome analysis of P. megaterium G18 revealed several key genes associated with acid tolerance. Targeted inactivation of the ydaG gene from SigB regulon, a general stress response gene, significantly reduced growth under acidic conditions compared to the wild type. qRT-PCR analysis showed increased ydaG expression in acidic conditions, further supporting its role in acid stress response. Microscopic analysis revealed no morphological differences between wild-type and mutant cells, suggesting that ydaG is not involved in maintaining cellular morphology but in facilitating acid tolerance through stress protein production. This research contributes to understanding the molecular mechanisms underlying acid tolerance in soil bacteria, P. megaterium, shedding light on potential applications in agriculture and industry.

Isolation and molecular characterization of fowl adenovirus from inclusion body hepatitis cases in Western India during 2019-2021.

Inclusion body hepatitis (IBH) resulted in a substantial economic loss in Western India during 2019 to 2021. The study aimed to characterize fowl adenovirus (FAdV) from field outbreaks. The study was conducted on 290 liver samples from 66 poultry flocks. The samples were subjected to histopathology and molecular detection, followed by phylogenetic typing of the partial hexon gene of the virus. Spiking mortality (14%) was recorded from day 21 to day 35 with peak mortality at the 28th day of age. The necropsy showed a pale and enlarged liver with hemorrhagic and yellowish necrotic foci, accumulation of straw-colored transudate in the pericardial sac which resulted in a flabby appearance of the heart, heart enlargement, and hemorrhages on the spleen, enlarged and congested kidneys. The virus inoculation resulted in stunting and poor feathering with hepatomegaly, hemorrhages and yellowish necrotic foci on the liver as well as greenish discoloration, and kidney swelling in SPF embryonated chicken eggs. Out of 29, 16 liver samples yielded 1219 bp amplicons specific to hexon gene fragments. The sequence and phylogenetic analysis identified 14 isolates as FAdV species E serotype 11 and two as species D serotype 8b. Conclusion: The results indicated that FAdV-8b and FAdV-11 strains are involved in disease outbreaks in western India.

Isolation and Characterization of Cefotaxime and Ciprofloxacin Co-Resistant Escherichia coli in Retail Chicken Carcasses

Transmission of antimicrobial-resistant bacteria to humans through the food chain is of great importance for public health. In this study, it was aimed to isolate and characterize the cefotaxime and ciprofloxacin-resistant Escherichia coli in retail chicken meat samples sold in Hatay. The isolates were subjected to phylogenetic group typing and antimicrobial susceptibility testing. The genetic relatedness of the isolates was determined using Enterobacterial Repetitive Intergenic Consensus Polymerase Chain Reaction (ERIC-PCR) technique. The isolates were also screened for the presence of both antimicrobial and plasmid-mediated quinolone resistance (PMQR) genes by PCR. Cefotaxime and ciprofloxacin co-resistant E. coli isolates with diverse genetic origins were recovered in 42.3% (22/52) of retail chicken carcasses. The E. coli isolates belonged to the phylogenetic group D2 were dominant (40.9%, 9/22), followed by B1 (27.3%, 6/22), B23 (18.2%, 4/22), and A1 (13.6%, 3/22), respectively. Based on dendrogram analysis, the ERIC-PCR method differentiated the isolates into 10 clusters (I-X). The multidrug resistance (MDR) was observed in 81.8% (18/22) of the isolates. PMQR determinants were not identified in any isolates tested. Molecular analysis revealed one or more β-lactamase-encoding genes in all isolates as a single or in combination: blaCTX-M-blaTEM (n=5), blaCMY-2 (n=5), blaCTX-M (n=5), blaCMY-2-blaSHV (n=3), blaCMY-2-blaTEM (n=3), and blaCTX-M-blaCMY-2 (n=1). This study highlights that retail chicken meat is an important reservoir of cefotaxime and ciprofloxacin co-resistant E. coli isolates. It is necessary to evaluate their contribution to the community and hospital infections.

Molecular epidemiology and characterization of antimicrobial-resistant Staphylococcus haemolyticus strains isolated from dairy cattle milk in Northwest, China.

Non-aureus Staphylococcus (NAS) species are currently the most commonly identified microbial agents causing sub-clinical infections of the udder and are also deemed as opportunistic pathogens of clinical mastitis in dairy cattle. More than 10 NAS species have been identified and studied but little is known about S. haemolyticus in accordance with dairy mastitis. The present study focused on the molecular epidemiology and genotypic characterization of S. haemolyticus isolated from dairy cattle milk in Northwest, China. In this study, a total of 356 milk samples were collected from large dairy farms in three provinces in Northwest, China. The bacterial isolation and presumptive identification were done by microbiological and biochemical methods following the molecular confirmation by 16S rRNA gene sequencing. The antimicrobial susceptibility testing (AST) was done by Kirby-Bauer disk diffusion assay and antibiotic-resistance genes (ARGs) were identified by PCR. The phylogenetic grouping and sequence typing was done by Pulsed Field Gel Electrophoresis (PFGE) and Multi-Locus Sequence Typing (MLST) respectively. In total, 39/356 (11.0%) were identified as positive for S. haemolyticus. The overall prevalence of other Staphylococcus species was noted to be 39.6% (141/356), while the species distribution was as follows: S. aureus 14.9%, S. sciuri 10.4%, S. saprophyticus 7.6%, S. chromogenes 4.2%, S. simulans 1.4%, and S. epidermidis 1.1%. The antimicrobial susceptibility of 39 S. haemolyticus strains exhibited higher resistance to erythromycin (92.3%) followed by trimethoprim-sulfamethoxazole (51.3%), ciprofloxacin (43.6%), florfenicol (30.8%), cefoxitin (28.2%), and gentamicin (23.1%). All of the S. haemolyticus strains were susceptible to tetracycline, vancomycin, and linezolid. The overall percentage of multi-drug resistant (MDR) S. haemolyticus strains was noted to be 46.15% (18/39). Among ARGs, mphC was identified as predominant (82.05%), followed by ermB (33.33%), floR (30.77%), gyrA (30.77%), sul1 (28.21%), ermA (23.08%), aadD (12.82%), grlA (12.82%), aacA-aphD (10.26%), sul2 (10.26%), dfrA (7.69%), and dfrG (5.13%). The PFGE categorized 39 S. haemolyticus strains into A-H phylogenetic groups while the MLST categorized strains into eight STs with ST8 being the most predominant while other STs identified were ST3, ST11, ST22, ST32, ST19, ST16, and ST7. These findings provided new insights into our understanding of the epidemiology and genetic characteristics of S. haemolyticus in dairy farms to inform interventions limiting the spread of AMR in dairy production.

Core genome multilocus sequence typing scheme for Bacillus cereus group bacteria

Core genome multilocus sequence typing (cgMLST) employs a strategy where the set of orthologous genes common to all members of a group of organisms are used for phylogenetic analysis of the group members. The Bacillus cereus group consists of species with pathogenicity towards insect species as well as warm-blooded animals including humans. While B. cereus is an opportunistic pathogen linked to a range of human disease conditions, including emesis and diarrhoea, Bacillus thuringiensis is an entomopathogenic species with toxicity toward insect larvae, and therefore used as a biological pesticide worldwide. Bacillus anthracis is a classical obligate pathogen causing anthrax, an acute lethal condition in herbivores as well as humans, and which is endemic in many parts of the world. The group also includes a range of additional species, and B. cereus group bacteria have been subject to analysis with a wide variety of phylogenetic typing systems. Here we present, based on analyses of 173 complete genomes from B. cereus group species available in public databases, the identification of a set of 1568 core genes which were used to create a core genome multilocus typing scheme for the group which is implemented in the PubMLST system as an open online database freely available to the community. The new cgMLST system provides unprecedented resolution over existing phylogenetic analysis schemes covering the B. cereus group.

Detection and characterization of hybrid uropathogenic Escherichia coli strains among E. coli isolates causing community-acquired urinary tract infection.

Introduction. The main aetiological agent of urinary tract infection (UTI) is Escherichia coli, categorized as uropathogenic E. coli (UPEC). The genome of UPEC shows a high degree of plasticity, which leads to the emergence of 'intermediary strains' with different traits from the parental pathotypes.Gap Statement/Aim. We aimed to assess the frequency and types of the hybrid UPEC among isolates causing UTI and characterize virulence properties of these hybrid isolates molecularly and phenotypically.Methodology. After detection of intestinal pathogenic E. coli (IPEC) virulence markers among 200 UPEC isolates, they were assessed for the presence of 40 virulence genes (VGs) of extraintestinal, uropathogenic and diarrhoeagenic E. coli, phylogenetic group typing, phenotypic traits including biofilm formation, adherence and invasion to HeLa cells, haemolysis activity and antimicrobial resistance.Results. The analysis showed 21 (10.5 %) UPEC isolates carried enteroaggregative E. coli (EAEC) and enteropathogenic E. coli (EPEC) virulence markers. Twenty isolates carried the aggR (EAEC) and one the eae and escV genes (EPEC), which were classified as hybrid strains. The most commonly identified genes were fimH (71.5 %), fyuA (66.7 %), iutA (62 %), chuA (57.1) and traT (47.6 %). Biofilm production, adhesion and invasion were found among 17 (81), 18 (85.7) and 11 (52.4 %) hybrids, respectively. Investigation of the genetic characteristics, phylogenetic group and virulence profile of the detected hybrids revealed that they have genetic diversity and do not belong to a particular clonal lineage.Conclusion. The present study reveals that some UPEC may carry virulence markers of IPEC pathotypes. EAEC and EPEC seem to have a greater tendency to form hybrids and cause UTI. Further studies are needed to elucidate what factors contributed to survival in the urinary tract system and facilitate infection and whether these combinations lead to an increase in pathogenicity or not.

Isolation and molecular characterization of infectious bursal disease virus circulating in western and central India.

Infectious bursal disease (IBD) caused substantial economic loss in central and western India during 2020 and 2021. The study was conducted to characterize IBD virus (IBDV) from field outbreaks. The study was conducted on 360 samples from 42 poultry flocks. The samples were subjected to histopathology and molecular detection, followed by phylogenetic typing of the partial VP2 gene. The mortality ranged from 15.25 to 60.18%. The necropsy showed hemorrhages on thigh muscles, mottled spleen, swelling of kidneys and bursae with hemorrhages, and cheesy exudate. Histopathology revealed extensive necrosis and depletion of follicular lymphoid cells within the cortex and medulla, along with widespread hemorrhages, edema, and cystic cavities in the bursa. The field isolates showed cytopathic effects in the seventh passage. The cytopathic effects included swelling, rounding, granulation of cytoplasm around the nucleus, fragmentation of the infected cells, and detachment. The reverse transcription-polymerase chain reaction amplified 664 bp partial VP2 gene. The phylogenetic analysis identified 19 field isolates as very virulent IBDVs (vvIBDVs) and three as classical strains. The results indicated that different vvIBDV strains are involved in disease outbreaks in central and western India.

309. Genetic Diversity and Conservation of Envelope Gene of SARS-CoV-2 and its Variants B.1.1.7, B.1.351, P.1, B.1.617.2 and B.1.1.529 with comparison with other human coronaviruses

Abstract Background SARS-CoV-2 E gene PCRs have been widely used as the first-line assay with a higher sensitivity than those targeting N or RdRp gene. Given the currently available primers and probes were designed at the onset of the pandemic, it is unknown whether the SARS-CoV-2 VOCs have accumulated significant mutations that may affect E gene PCRs. In this study we aim to perform a comprehensive genetic analysis of SARS-CoV-2 E gene sequences to evaluate the impact of the emerging VOCs on E gene PCR performance. Methods 600 whole-genome sequences of 7 species of human coronaviruses (HCoVs) were retrieved from GenBank and GISAID, including Sarbecoviruses (SARS-CoV-2 variants B1.1.7, B1.351, P.1, B.1.617.2 and B.1.1.529, and SARS-CoV), Embecovirus (OC43, HKU1), Merbecovirus (MERS) and Alphacoronaviruses (229E, NL63). The E gene sequences were retrieved from full-length genomes of corresponding viruses and aligned by ClustalW multiple alignment. Phylogenetic, conservation and mutation analyses analysis of the enrolled sequences was performed. Results E gene-based phylogenetic analysis yielded HCoVs typing results consistent with whole genome typing, suggesting E gene is a reliable locus for phylogenetic analysis and typing of HCoVs. Four pan-Sarbecovirus conserved E gene regions were identified with 100% conserved nucleotide similarity among SARS-CoV-2 and its VOCs, as well as SARS-CoV. These regions have appropriate G/C content which may be suitable for primer/probe design for E gene-based pan-Sarbecovirus screening assay. No significant E mutations were found in 137 retrieved SARS-CoV-2 and its VOCs. Interestingly, two novel variations, C26299U and T26354A, were identified in two of our SARS-CoV-2 strains. The latter variation occurred at the 3’ end of the target region of the widely used Charité/Berlin (WHO) probe. This variant may lead to a potential failure of the first-line E gene PCR. Conclusion Our data shed light on the genetic diversity and conservation of E gene of SARS-CoV-2 and may be beneficial for future primer/probe design for novel first-line assay or SARS-CoV-2-specific E gene PCR. SARS-CoV-2 VOCs have not accumulated significant mutations in E gene so far. The impact of novel E gene variations C26299U and T26354A on molecular diagnostic testing warrants further investigation. Disclosures All Authors: No reported disclosures.

A Practical Bioinformatics Workflow for Routine Analysis of Bacterial WGS Data.

The use of whole-genome sequencing (WGS) for bacterial characterisation has increased substantially in the last decade. Its high throughput and decreasing cost have led to significant changes in outbreak investigations and surveillance of a wide variety of microbial pathogens. Despite the innumerable advantages of WGS, several drawbacks concerning data analysis and management, as well as a general lack of standardisation, hinder its integration in routine use. In this work, a bioinformatics workflow for (Illumina) WGS data is presented for bacterial characterisation including genome annotation, species identification, serotype prediction, antimicrobial resistance prediction, virulence-related genes and plasmid replicon detection, core-genome-based or single nucleotide polymorphism (SNP)-based phylogenetic clustering and sequence typing. Workflow was tested using a collection of 22 in-house sequences of Salmonella enterica isolates belonging to a local outbreak, coupled with a collection of 182 Salmonella genomes publicly available. No errors were reported during the execution period, and all genomes were analysed. The bioinformatics workflow can be tailored to other pathogens of interest and is freely available for academic and non-profit use as an uploadable file to the Galaxy platform.

The prevalence and characteristics of extended-spectrum β-lactamase Escherichia coli in raw milk and dairy farms in Northern Xinjiang, China

In this study, extended-spectrum β-lactamase (ESBL) Escherichia coli were isolated from five dairy farms in three areas of northern Xinjiang, China. Molecular biological techniques were used to systematically analyze drug resistance phenotypes and genotypes, virulence genes, phylogenetics, biofilm formation (BF), and pulsed field gel electrophoresis (PFGE) typing of isolated ESBL E. coli strains. A total of 766 samples were collected from five dairy farms in Shihezi, Urumqi and Yili, from which 149 (19.5 %, 95 % CI: 16.65 %–22.25 %) ESBL E. coli strains were isolated. Their distribution and contamination levels varied from region to region, with 16.2 % (68/419) in Urumqi, 22.4 % (60/268) in Shihezi, and 26.6 % (21/79) in Yili. The majority of isolates (97.3 %, 145/149) harbored the β-lactamase blaCTX-M gene; while blaCTX-M-1 was the dominant phylogenetic group. The analysis of 21 resistance genes and the susceptibility to 13 different antibiotics showed that 91.3 % (136/149) of strains were resistant to three or more antibiotics. Thirty-six strains (24.2 %) belonged to extraintestinal pathogenic E. coli (ExPEC), and phylogenetic typing results were mainly grouped A (50.3 %) and B1 (37.6 %). Also, the biofilm assay revealed that 112 strains (75.2 %) could form biofilms. PFGE results showed that the 49 isolates revealed 21 major pulsotypes (P1–P21) and 28 subtypes with 80 % similarity, indicating the overall genetic diversity in the distribution area and sources of the samples.

Physiology, metabolism, antibiotic resistance, and genetic diversity of Harveyi clade bacteria isolated from coastal mariculture system in China in the last two decades

Vibrio bacteria, particularly members of the Harveyi clade, are the most important pathogens of aquatic organisms that cause significant economic losses in the world. It is difficult to provide specific data on taxa of the Harveyi clade for biological research and prevention strategies. Therefore, we conducted an extensive phenotypic and antibiotic resistance study, as well as phylogenetic and molecular typing of 192 isolates of the Harveyi clade collection from 2000 to 2020 with a typical interannual difference from a coastal area in China. The isolates had a significant interspecific genetic and antibiotic resistance diversity. Based on the multilocus sequence analysis (MLSA) of housekeeping genes (gyrB, pyrH, recA, and atpA), 192 Harveyi clade isolates were rapidly and accurately classified into 10 species. The population of these isolates was composed of 95 sequence types (STs), of which 92 STs were newly identified, indicating a high degree of genetic diversity. ST327 ranked first, accounting for 11.5% of the total number of isolates (22 out of 192), followed by ST215 with 6.25%, while 63 STs included single isolates. At the metabolic level, the physiological and biochemical experiments revealed that all the Harveyi clade isolates were positive for oxidase and negative for melibiose. The isolates showed a varied tolerance to 11 antibiotics. No isolates were resistant to neomycin. The percentages of sulfadimidine-resistant strains (61 out of 192), sulfadiazine (44 out of 192), sulfamonomethoxine (44 out of 192), sulfamethoxazole (33 out of 192), thiamphenicol (34 out of 192), ciprofloxacin (52 out of 192), and enrofloxacin (31 out of 192) were 31.77%, 22.92%, 22.92%, 17.19%, 17.71%, 27.08%, and 16.15%, respectively. A proportion of 61.8% of the isolates presented a multiple antibiotic resistance index (MARI) lower than 0.1, indicating that the risk of antibiotic resistance transmission of most of the Harveyi clade is low in mariculture systems in China. These results provide substantial data to support further studies on the identification and genetic and metabolic diversity of Harveyi clade isolates in mariculture systems in China.

Changes in the characteristics of community-onset fluoroquinolone-resistant Escherichia coli isolates causing community-acquired acute pyelonephritis in South Korea.

This study aimed to examine the changes in the characteristics of community-onset fluoroquinolone-resistant (FQ-R) Escherichia coli isolates causing community-acquired acute pyelonephritis (APN) in South Korea. Blood or urine samples were prospectively collected from patients aged ≥15 years with community-acquired APN who were admitted to one of the eight Korean hospitals included in this study between September 2017 and August 2018. Phylogenetic typing, multilocus sequence typing, and molecular characterization of β-lactamase resistance and plasmid-mediated quinolone resistance (PMQR) determinants were performed. The data were compared with those from a previous study with the same design conducted in 2010-2011. A total of 300 and 346 isolates were identified in 2010-2011 and 2017-2018, respectively. Among them, 76 (22.0%) and 77 (25.7%) FQ-R isolates were identified in 2010-2011 and 2017-2018, respectively. A significantly higher antimicrobial resistance against third-to fourth-generation cephalosporins, including cefotaxime (23.9% vs. 77.9%, P<0.001), were observed among FQ-R isolates in 2017-2018 than among those in 2010-2011. A higher proportion of ST131 isolates (27.6% vs. 66.2%, P<0.001), as well as isolates that had extended-spectrum β-lactamase (ESBL)/plasmid-mediated AmpC β-lactamase (PABL) (23.7% vs. 79.2%, P<0.001), was observed in 2017-2018 than in 2010-2011. Further, more PMQR determinants (11.8% vs. 40.8%, P<0.001) were observed in 2017-2018 than in 2010-2011. Among uropathogenic FQ-R E. coli isolates in South Korea, the prevalence of ST131 and the proportion of isolates containing ESBL and/or PMQR determinants have increased.

Assembly of a Large Collection of Maxicircle Sequences and Their Usefulness for Leishmania Taxonomy and Strain Typing.

Parasites of medical importance, such as Leishmania and Trypanosoma, are characterized by the presence of thousands of circular DNA molecules forming a structure known as kinetoplast, within the mitochondria. The maxicircles, which are equivalent to the mitochondrial genome in other eukaryotes, have been proposed as a promising phylogenetic marker. Using whole-DNA sequencing data, it is also possible to assemble maxicircle sequences as shown here and in previous works. In this study, based on data available in public databases and using a bioinformatics workflow previously reported by our group, we assembled the complete coding region of the maxicircles for 26 prototypical strains of trypanosomatid species. Phylogenetic analysis based on this dataset resulted in a robust tree showing an accurate taxonomy of kinetoplastids, which was also able to discern between closely related Leishmania species that are usually difficult to discriminate by classical methodologies. In addition, we provide a dataset of the maxicircle sequences of 60 Leishmania infantum field isolates from America, Western Europe, North Africa, and Eastern Europe. In agreement with previous studies, our data indicate that L. infantum parasites from Brazil are highly homogeneous and closely related to European strains, which were transferred there during the discovery of America. However, this study showed the existence of different L. infantum populations/clades within the Mediterranean region. A maxicircle signature for each clade has been established. Interestingly, two L. infantum clades were found coexisting in the same region of Spain, one similar to the American strains, represented by the Spanish JPCM5 reference strain, and the other, named “non-JPC like”, may be related to an important leishmaniasis outbreak that occurred in Madrid a few years ago. In conclusion, the maxicircle sequence emerges as a robust molecular marker for phylogenetic analysis and species typing within the kinetoplastids, which also has the potential to discriminate intraspecific variability.

Antibiotic resistance, phylogenetic typing, and virulence genes profile analysis of uropathogenic Escherichia coli isolated from patients in southern Iraq.

Of the most common infectious diseases that occur mainly by uropathogenic Escherichia coli (UPEC) is urinary tract infections (UTIs). The purpose of this study was to investigate virulence factors, antibiotic resistance, and phylogenetic groups among UPEC strains isolated from patients with UTI in southern Iraq. A total of 100 UPEC isolates were collected from urine samples of UTI patients from various hospitals in southern Iraq, and confirmed by morphological and biochemical tests. Antimicrobial susceptibility testing on isolates was performed by disk diffusion method. Multiplex PCR techniques were used to evaluate the phylogenetic groups based on Clermont method and to detect the presence of six virulence factor genes. The majority of isolates belonged to the phylogenetic groups B2 (46%) and C (13%). The most prevalent virulence factors were fimH (96%), followed by aer (47%), papC (36%), cnf1 (17%), hly (15%), and afa (8%). Phenotypic testing showed that the isolates were most resistant to piperacillin, ticarcillin, amoxicillin/clavulanic acid (92%, 91%, and 88%, respectively) and most sensitive to amikacin and imipenem, respectively. The maximum antibiotic resistance and virulence factors were observed in the phylogenetic group B2. The results showed that the UPEC isolates had all six virulence factors with high frequency and the highest drug resistance. Besides, the results showed a direct relationship between virulence factors, gene diversity, phylogenetic background, and antimicrobial resistance in the UPEC isolates.