Clinical and Bacteriological Specificities of Escherichia coli Bloodstream Infections From Biliary Portal of Entries.

BACKGROUND: Escherichia coli is frequently responsible for bloodstream infections (BSIs). Among digestive BSIs, biliary infections appear to be less severe. Respective roles of host factors, bacterial determinants (phylogroups, virulence, and antibiotic resistance), and portal of entry on outcome are unknown. METHODS: Clinical characteristics and prognosis of 770 episodes of E coli BSI were analyzed and isolates sequenced (Illumina technology) comparing phylogroups, multilocus sequence type, virulence, and resistance gene content. BSI isolates were compared with 362 commensal E coli from healthy subjects. RESULTS: Among 770 episodes, 135 were biliary, 156 nonbiliary digestive, and 479 urinary. Compared to urinary infections, BSIs of digestive origin occurred significantly more in men, comorbid, and immunocompromised patients. Digestive portal of entry was significantly associated with septic shock and death. Among digestive infections, patients with biliary infections were less likely to die (P = .032), despite comparable initial severity. Biliary E coli resembled commensals (phylogroup distribution, sequence type, and few virulence-associated genes) whereas nonbiliary digestive and urinary strains carried many virulence-associated genes. CONCLUSIONS: Escherichia coli strains responsible for biliary infections exhibit commensal characteristics and are associated with lower mortality rates, despite similar initial severity, than other digestive BSIs. Biliary drainage in addition to antibiotics in the management of biliary infections may explain improved outcome.

Antimicrobial resistance and heterogeneity of Neisseria gonorrhoeae isolated from patients attending sexually transmitted infection clinics in Lusaka, Zambia.

BACKGROUND: Antimicrobial resistance (AMR) of Neisseria gonorrhoeae is a threat to public health as strains have developed resistance to antimicrobials available for the treatment of gonorrhea. Whole genome sequencing (WGS) can detect and predict antimicrobial resistance to enhance the control and prevention of gonorrhea. Data on the molecular epidemiology of N. gonorrhoeae is sparse in Zambia. This study aimed to determine the genetic diversity of N. gonorrhoeae isolated from patients attending sexually transmitted infection (STI) clinics in Lusaka, Zambia. METHODS: A cross-sectional study that sequenced 38 N. gonorrhoeae isolated from 122 patients with gonorrhea from 2019 to 2020 was conducted. The AMR profiles were determined by the E-test, and the DNA was extracted using the NucliSens easyMaG magnetic device. Whole genome sequencing was performed on the Illumina NextSeq550 platform. The Bacterial analysis pipeline (BAP) that is readily available at: https://cge.cbs.dtu.dk/services/CGEpipeline-1.1 was used for the identification of the species, assembling the genome, multi-locus sequence typing (MLST), detection of plasmids and AMR genes. Phylogeny by single nucleotide polymorphisms (SNPs) was determined with the CCphylo dataset. RESULTS: The most frequent STs with 18.4% of isolates each were ST7363, ST1921 and ST1582, followed by ST1583 (13%), novel ST17026 (7.9%), ST1588 (7.9%), ST1596 (5.3%), ST11181 (5.3%), ST11750 (2.6/%) and ST11241 (2.6%) among the 38 genotyped isolates. The blaTeM-1B and tetM (55%) was the most prevalent combination of AMR genes, followed by blaTeM-1B (18.4%), tetM (15.8%), and the combination of blaTeM-1B, ermT, and tetL was 2.6% of the isolates. The AMR phenotypes were predicted in ciprofloxacin, penicillin, tetracycline, azithromycin, and cefixime. The combination of mutations 23.7% was gryA (S91F), parC (E91G), ponA (L421) and rpsJ (V57M), followed by 18.4% in gyrA (S91F), ponA (L421P), rpsJ (V57M), and 18.4% in gyrA (D95G, S91F), ponA (L421P), and rpsJ (V57M). The combinations in gyrA (D95G, S91F) and rpsJ (V57M), and gyrA (D95G, S91F), parC (E91F), ponA (L421P) and rpsJ (V57M) were 13.2% each of the isolates. Plasmid TEM-1 (84.2%), tetM (15.8%), and gonococcal genetic island (GGI) was detected in all isolates. CONCLUSION: This study revealed remarkable heterogeneity of N. gonorrhoeae with blaTEM-1, tetM, ponA, gyrA, and parC genes associated with high resistance to penicillin, tetracycline, and ciprofloxacin demanding revision of the standard treatment guidelines and improved antimicrobial stewardship in Zambia.

Microbiome and mitogenomics of the chigger mite Pentidionis agamae: potential role as an Orientia vector and associations with divergent clades of Wolbachia and Borrelia.

BACKGROUND: Trombiculid mites are globally distributed, highly diverse arachnids that largely lack molecular resources such as whole mitogenomes for the elucidation of taxonomic relationships. Trombiculid larvae (chiggers) parasitise vertebrates and can transmit bacteria (Orientia spp.) responsible for scrub typhus, a zoonotic febrile illness. Orientia tsutsugamushi causes most cases of scrub typhus and is endemic to the Asia-Pacific Region, where it is transmitted by Leptotrombidium spp. chiggers. However, in Dubai, Candidatus Orientia chuto was isolated from a case of scrub typhus and is also known to circulate among rodents in Saudi Arabia and Kenya, although its vectors remain poorly defined. In addition to Orientia, chiggers are often infected with other potential pathogens or arthropod-specific endosymbionts, but their significance for trombiculid biology and public health is unclear. RESULTS: Ten chigger species were collected from rodents in southwestern Saudi Arabia. Chiggers were pooled according to species and screened for Orientia DNA by PCR. Two species (Microtrombicula muhaylensis and Pentidionis agamae) produced positive results for the htrA gene, although Ca. Orientia chuto DNA was confirmed by Sanger sequencing only in P. agamae. Metagenomic sequencing of three pools of P. agamae provided evidence for two other bacterial associates: a spirochaete and a Wolbachia symbiont. Phylogenetic analysis of 16S rRNA and multi-locus sequence typing genes placed the spirochaete in a clade of micromammal-associated Borrelia spp. that are widely-distributed globally with no known vector. For the Wolbachia symbiont, a genome assembly was obtained that allowed phylogenetic localisation in a novel, divergent clade. Cytochrome c oxidase I (COI) barcodes for Saudi Arabian chiggers enabled comparisons with global chigger diversity, revealing several cases of discordance with classical taxonomy. Complete mitogenome assemblies were obtained for the three P. agamae pools and almost 50 SNPs were identified, despite a common geographic origin. CONCLUSIONS: P. agamae was identified as a potential vector of Ca. Orientia chuto on the Arabian Peninsula. The detection of an unusual Borrelia sp. and a divergent Wolbachia symbiont in P. agamae indicated links with chigger microbiomes in other parts of the world, while COI barcoding and mitogenomic analyses greatly extended our understanding of inter- and intraspecific relationships in trombiculid mites.

Newly Recognized Spotted Fever Group Rickettsia as Cause of Severe Rocky Mountain Spotted Fever-Like Illness, Northern California, USA.

The incidence of spotted fever group (SFG) rickettsioses in the United States has tripled since 2010. Rocky Mountain spotted fever, the most severe SFG rickettsiosis, is caused by Rickettsia rickettsii. The lack of species-specific confirmatory testing obfuscates the relative contribution of R. rickettsii and other SFG Rickettsia to this increase. We report a newly recognized rickettsial pathogen, Rickettsia sp. CA6269, as the cause of severe Rocky Mountain spotted fever-like illness in 2 case-patients residing in northern California. Multilocus sequence typing supported the recognition of this pathogen as a novel Rickettsia genotype most closely related to R. rickettsii. Cross-reactivity observed for an established molecular diagnostic test indicated that Rickettsia sp. CA6269 might be misidentified as R. rickettsii. We developed a Rickettsia sp. CA6269-specific real-time PCR to help resolve this diagnostic challenge and better characterize the spectrum of clinical disease and ecologic epidemiology of this pathogen.

C-terminal deletion of RelA protein is suggested as a possible cause of infective endocarditis recurrence with Enterococcus faecium.

Infective endocarditis (IE) caused by Enterococcus spp. represents the third most common cause of IE, with high rates of relapse compared with other bacteria. Interestingly, late relapses (>6 months) have only been described in Enterococcus faecalis, but here we describe the first reported IE relapse with Enterococcus faecium more than a year (17 months) after the initial endocarditis episode. Firstly, by multi locus sequence typing (MLST), we demonstrated that both isolates (EF646 and EF641) belong to the same sequence type (ST117). Considering that EF641 was able to overcome starvation and antibiotic treatment conditions surviving for a long period of time, we performed bioinformatic analysis in identifying potential genes involved in virulence and stringent response. Our results showed a 13-nucleotide duplication (positions 1638-1650) in the gene relA, resulting in a premature stop codon, with a loss of 167 amino acids from the C-terminal domains of the RelA enzyme. RelA mediates the stringent response in bacteria, modulating levels of the alarmone guanosine tetraphosphate (ppGpp). The relA mutant (EF641) was associated with lower growth capacity, the presence of small colony variants, and higher capacity to produce biofilms (compared with the strain EF646), but without differences in antimicrobial susceptibility patterns according to standard procedures during planktonic growth. Instead, EF641 demonstrated tolerance to high doses of teicoplanin when growing in a biofilm. We conclude that all these events would be closely related to the long-term survival of the E. faecium and the late relapse of the IE. These data represent the first clinical evidence of mutations in the stringent response (relA gene) related with E. faecium IE relapse.

Genomic analysis of inter-hospital transmission of vancomycin-resistant Enterococcus faecium sequence type 80 isolated during an outbreak in Hiroshima, Japan.

Outbreaks caused by vancomycin-resistant enterococci that transcend jurisdictional boundaries are occurring worldwide. This study focused on a vancomycin-resistant enterococcus outbreak that occurred between 2018 and 2021 across two cities in Hiroshima, Japan. The study involved genetic and phylogenetic analyses using whole-genome sequencing of 103 isolates of vancomycin-resistant enterococci to identify the source and transmission routes of the outbreak. Phylogenetic analysis was performed using core genome multilocus sequence typing and core single-nucleotide polymorphisms; infection routes between hospitals were inferred using BadTrIP. The outbreak was caused by Enterococcus faecium sequence type (ST) 80 carrying the vanA plasmid, which was derived from strain A10290 isolated in India. Of the 103 isolates, 93 were E. faecium ST80 transmitted across hospitals. The circular vanA plasmid of the Hiroshima isolates was similar to the vanA plasmid of strain A10290 and transferred from E. faecium ST80 to other STs of E. faecium and other Enterococcus species by conjugation. The inferred transmission routes across hospitals suggest the existence of a central hospital serving as a hub, propagating vancomycin-resistant enterococci to multiple hospitals. Our study highlights the importance of early intervention at the key central hospital to prevent the spread of the infection to small medical facilities, such as nursing homes, with limited medical resources and a high number of vulnerable individuals.

Characterization of MLST-99 Salmonella Typhimurium and the monophasic variant I:4,[5],12:i:- isolated from Canadian Atlantic coast shellfish.

Salmonella enterica subsp. enterica Typhimurium and its monophasic variant I 1;4,[5],12:i:- (MVST) are responsible for thousands of reported cases of salmonellosis each year in Canada, and countries worldwide. We investigated S. Typhimurium and MVST isolates recovered from raw shellfish harvested in Atlantic Canada by the Canadian Food Inspection Agency (CFIA) over the past decade, to assess the potential impact of these isolates on human illness and to explore possible routes of shellfish contamination. Whole-genome sequence analysis was performed on 210 isolates of S. Typhimurium and MVST recovered from various food sources, including shellfish. The objective was to identify genetic markers linked to ST-99, a sequence type specifically associated with shellfish, which could explain their high prevalence in shellfish. We also investigated the genetic similarity amongst CFIA ST-99 isolates recovered in different years and geographical locations. Finally, the study aimed to enhance the molecular serotyping of ST-99 isolates, as they are serologically classified as MVST but are frequently misidentified as S. Typhimurium through sequence analysis. To ensure recovery of ST-99 from shellfish was not due to favourable growth kinetics, we measured the growth rates of these isolates relative to other Salmonella and determined that ST-99 did not have a faster growth rate and/or shorter lag phase than other Salmonella evaluated. The CFIA ST-99 isolates from shellfish were highly clonal, with up to 81 high-quality single nucleotide variants amongst isolates. ST-99 isolates both within the CFIA collection and those isolated globally carried numerous unique deletions, insertions and mutations in genes, including some considered important for virulence, such as gene deletions in the type VI secretion system. Interestingly, several of these genetic characteristics appear to be unique to North America. Most notably was a large genomic region showing a high prevalence in genomes from Canadian isolates compared to those from the USA. Although the functions of the majority of the proteins encoded within this region remain unknown, the genes umuC and umuD, known to be protective against UV light damage, were present. While this study did not specifically examine the effects of mutations and insertions, results indicate that these isolates may be adapted to survive in specific environments, such as ocean water, where wild birds and/or animals serve as the natural hosts. Our hypothesis is reinforced by a global phylogenetic analysis, which indicates that isolates obtained from North American shellfish and wild birds are infrequently connected to isolates from human sources. These findings suggest a distinct ecological niche for ST-99, potentially indicating their specialization and adaptation to non-human hosts and environments, such as oceanic habitats.

CRISPR and CRISPR-MVLST reveal conserved spacer distribution and high similarity among Salmonella enterica serovar Infantis genomes from Brazil and other countries.

Salmonella enterica serovar Infantis (S. Infantis) is a globally distributed non-typhoid serovar infecting humans and food-producing animals. Considering the zoonotic potential and public health importance of this serovar, strategies to characterizing, monitor and control this pathogen are of great importance. This study aimed to determine the genetic relatedness of 80 Brazilian S. Infantis genomes in comparison to 40 non-Brazilian genomes from 14 countries using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-Multi-Locus Virulence Sequence Typing (CRISPR-MVLST). CRISPR spacers were searched using CRISPR-Cas++ and fimH and sseL alleles using BLAST and MEGA X. Results were analyzed using BioNumerics 7.6 in order to obtain similarity dendrograms. A total of 23 CRISPR1 and 11 CRISPR2 alleles formed by 37 and 26 types of spacers, respectively, were detected. MVLST revealed the presence of five fimH and three sseL alleles. CRISPR's similarity dendrogram showed 32 strain subtypes, with an overall similarity ≥ 78.6. The CRISPR-MVLST similarity dendrogram showed 37 subtypes, with an overall similarity ≥ 79.2. In conclusion, S. Infantis strains isolated from diverse sources in Brazil and other countries presented a high genetic similarity according to CRISPR and CRISPR-MVLST, regardless of their source, year, and/or place of isolation. These results suggest that both methods might be useful for molecular typing S. Infantis strains using WGS data.

Core genome multilocus sequence typing (cgMLST) applicable to the monophyletic Klebsiella oxytoca species complex.

The environmental bacterium Klebsiella oxytoca displays an alarming increase of antibiotic-resistant strains that frequently cause outbreaks in intensive care units. Due to its prevalence in the environment and opportunistic presence in humans, molecular surveillance (including resistance marker screening) and high-resolution cluster analysis are of high relevance. Furthermore, K. oxytoca previously described in studies is rather a species complex (KoSC) than a single species comprising at least six closely related species that are not easily differentiated by standard typing methods. To reach a discriminatory power high enough to identify and resolve clusters within these species, whole genome sequencing is necessary. The resolution is achievable with core genome multilocus sequence typing (cgMLST) extending typing of a few housekeeping genes to thousands of core genome genes. CgMLST is highly standardized and provides a nomenclature enabling cross laboratory reproducibility and data exchange for routine diagnostics. Here, we established a cgMLST scheme not only capable of resolving the KoSC species but also producing reliable and consistent results for published outbreaks. Our cgMLST scheme consists of 2,536 core genome and 2,693 accessory genome targets, with a percentage of good cgMLST targets of 98.31% in 880 KoSC genomes downloaded from the National Center for Biotechnology Information (NCBI). We also validated resistance markers against known resistance gene patterns and successfully linked genetic results to phenotypically confirmed toxic strains carrying the til gene cluster. In conclusion, our novel cgMLST enables highly reproducible typing of four different clinically relevant species of the KoSC and thus facilitates molecular surveillance and cluster investigations.

Multi-locus sequencing typing reveals geographically related intraspecies variability of Sporothrix brasiliensis.

Sporotrichosis is a subcutaneous mycosis caused by pathogenic Sporothrix species. Among them, Sporothrix brasiliensis is the main species associated with endemic regions in South America, especially Brazil. It is highly virulent and can be spread through zoonotic transmission. Molecular epidemiological surveys are needed to determine the extent of genetic variation, to investigate outbreaks, and to identify genotypes associated with antifungal resistance and susceptibility. This study investigated the sequence variation of different constitutive genes and established a novel multilocus sequence typing (MLST) scheme for S. brasiliensis. Specific primers were designed for 16 genes using Primer-BLAST software based on the genome sequences of three S. brasiliensis strains (ATCC MYA-4823, A001 and A005). Ninety-one human, animal, and environmental S. brasiliensis isolates from different Brazilian geographic regions (South, Southeast, Midwest and Northeast) andtwo isolates from Paraguay were sequenced. The loci that presented the highest nucleotide diversity (π) were selected for the MLST scheme. Among the 16 studied genetic loci, four presented increased π value and were able to distinguish all S. brasiliensis isolates into seven distinct haplotypes. The PCR conditions were standardized for four loci. Some of the obtained haplotypes were associated with the geographic origin of the strains. This study presents an important advance in the understanding of this important agent of sporotrichosis in Brazil. It significantly increased the discriminatory power for genotyping of S. brasiliensis isolates, and enabled new contributions to the epidemiological studies of this human and animal pathogen in Brazil and in other countries.

Multi-locus sequence typing and phylogenetics of Cryptococcus neoformans AD hybrids.

Hybrid AD strains of the human pathogenic Cryptococcus neoformans species complex have been reported from many parts of the world. However, their origin, diversity, and evolution are incompletely understood. In this study, we analyzed 102 AD hybrid strains representing 21 countries on five continents. For each strain, we obtained its mating type and its allelic sequences at each of the seven loci that have been used for genotyping haploid serotypes A and D strains of the species complex by the Cryptococcus research community. Our results showed that most AD hybrids exhibited loss of heterozygosity at one or more of the seven analyzed loci. Phylogenetic and population genetic analyses of the allelic sequences revealed multiple origins of the hybrids within each continent, dating back to one million years ago in Africa and up to the present in other continents. We found evidence for clonal reproduction and long-distance dispersal of these hybrids in nature. Comparisons with the global haploid serotypes A and D strains identified new alleles and new haploid multi-locus genotypes in AD hybrids, consistent with the presence of yet-to-be discovered genetic diversity in haploid populations of this species complex in nature. Together, our results indicate that AD hybrids can be effectively genotyped using the same multi-locus sequencing type approach as that established for serotypes A and D strains. Our comparisons of the AD hybrids among each other as well as with the global haploid serotypes A and D strains revealed novel genetic diversity as well as evidence for multiple origins and dynamic evolution of these hybrids in nature.

Clostridium neonatale antimicrobial susceptibility, genetic resistance determinants, and genotyping: a multicentre spatiotemporal retrospective analysis.

BACKGROUND: Clostridium neonatale was isolated during an outbreak of neonatal necrotizing enterocolitis (NEC) in 2002. C. neonatale was validated as a new species within the genus Clostridium sensu stricto in 2018. In the present study, we evaluated the antimicrobial susceptibility, genetic determinants of resistance, and phylogenetic relationships of a collection of clinical isolates of C. neonatale. METHODS: C. neonatale strains (n = 68) were isolated from the stools of preterm neonates who either developed NEC or were asymptomatic carriers of C. neonatale in different periods and in different hospitals. Antimicrobial susceptibility was determined by the disc diffusion method. The MICs of clindamycin, cefotaxime and tetracycline were determined. Genetic determinants of resistance were screened by PCR (n = 68) and WGS (n = 35). Genotyping of the isolates was performed by MLST. RESULTS: Antimicrobial resistance was found to clindamycin (n = 24; 35%), cefotaxime (n = 7; 10%) and tetracycline (n = 1; 1%). One clindamycin-resistant isolate carried erm(B) by PCR. In addition, one isolate carrying tet(M) was tetracycline resistant (MIC = 16 mg/L) and 44 isolates carrying either tet(O), tet(32) or tet(M) were tetracycline susceptible (MICs < 16 mg/L). MLST showed that ST2 and ST15 were significantly associated with tet(32) (P < 0.0001) and tet(O) (P < 0.0001), respectively. From WGS, we identified aph(3')-IIa and blaTEM-116 genes and a blaCBP-1-like gene. CONCLUSIONS: C. neonatale is susceptible to anti-anaerobic molecules but resistant to clindamycin, cefotaxime and tetracycline. Genes encoding tetracycline ribosomal protection, macrolide-lincosamide-streptogramin B rRNA methyltransferase, aminoglycoside 3'-phosphotransferase and ß-lactamases have been identified in genomic regions flanked by mobile genetic elements.

WGS of a cluster of MDR Shigella sonnei utilizing Oxford Nanopore R10.4.1 long-read sequencing.

OBJECTIVES: To utilize long-read nanopore sequencing (R10.4.1 flowcells) for WGS of a cluster of MDR Shigella sonnei, specifically characterizing genetic predictors of antimicrobial resistance (AMR). METHODS: WGS was performed on S. sonnei isolates identified from stool and blood between September 2021 and October 2022. Bacterial DNA from clinical isolates was extracted on the MagNA Pure 24 and sequenced on the GridION utilizing R10.4.1 flowcells. Phenotypic antimicrobial susceptibility testing was interpreted based on CLSI breakpoints. Sequencing data were processed with BugSeq, and AMR was assessed with BugSplit and ResFinder. RESULTS: Fifty-six isolates were sequenced, including 53 related to the cluster of cases. All cluster isolates were identified as S. sonnei by sequencing, with global genotype 3.6.1.1.2 (CipR.MSM5), MLST 152 and PopPUNK cluster 3. Core genome MLST (cgMLST, examining 2513 loci) and reference-based MLST (refMLST, examining 4091 loci) both confirmed the clonality of the isolates. Cluster isolates were resistant to ampicillin (blaTEM-1), trimethoprim/sulfamethoxazole (dfA1, dfrA17; sul1, sul2), azithromycin (ermB, mphA) and ciprofloxacin (gyrA S83L, gyrA D87G, parC S80I). No genomic predictors of resistance to carbapenems were identified. CONCLUSIONS: WGS with R10.4.1 enabled rapid sequencing and identification of an MDR S. sonnei community cluster. Genetic predictors of AMR were concordant with phenotypic antimicrobial susceptibility testing.

Use of Fourier-transform infrared spectroscopy for real-time outbreak investigation of OXA-48-producing Escherichia coli.

BACKGROUND: Efficient infection control during carbapenem-resistant Enterobacterales outbreaks demands rapid and simple techniques for outbreak investigations. WGS, the current gold standard for outbreak identification, is expensive, time-consuming and requires a high level of expertise. Fourier-transform infrared (FTIR) spectroscopy (IR Biotyper) is a rapid typing method based on infrared radiation applied to samples, which provides a highly specific absorption spectrum. OBJECTIVES: To investigate an outbreak of OXA-48-producing Escherichia coli in real-time using FTIR and subsequently compare the results with WGS. METHODS: Twenty-one isolates were collected during a nosocomial outbreak, and identification and antibiotic susceptibilities were confirmed by VITEK®2. FTIR was conducted for all isolates, and nine representative isolates were sequenced. RESULTS: FTIR was able to correctly determine the clonal relatedness of the isolates and to identify the outbreak cluster, as confirmed by WGS. By WGS, isolates in the main FTIR cluster belonged to the same MLST type and core-genome MLST type, and they harboured similar plasmids and resistance genes, whereas the singletons external to the FTIR cluster had different genetic content. CONCLUSIONS: FTIR can operate as a rapid, efficient and reliable first-line tool for outbreak investigations during a real-time ongoing E. coli outbreak, which can contribute to limiting the spread of pathogens.

Novel sequence type of carbapenem-resistant Acinetobacter pittii ST1451 with enhanced virulence isolated from septicaemic neonates in India.

BACKGROUND: The clinical relevance of Acinetobacter pittii is increasing, but reports of this organism causing neonatal sepsis are rare. OBJECTIVES: To understand the mechanisms of resistance and virulence of A. pittii isolated from neonatal blood belonging to a novel sequence type. MATERIALS AND METHODS: Antibiotic susceptibility, MLST, WGS, phylogenomic comparison with a global collection of carbapenemase-harbouring A. pittii were done. To study the pathogenic potential of novel A. pittii, in vitro and in vivo assays were carried out. RESULTS AND DISCUSSION: Two novel multidrug-resistant A. pittii from neonatal blood belonging to a novel sequence type 1451 (ST1451) were isolated. WGS revealed that the isolates were almost similar (147 SNP distant) and harbouring two carbapenem resistance genes blaNDM-1 with upstream ISAba125 and downstream bleMBL along with blaOXA-58 with upstream ISAba3. Other resistance genes included blaADC-25, blaOXA-533, aph(3″)-Ib, aph(3')-VIa, aph(6)-Id, aac(3)-IId, mph(E), msr(E), sul2 and tet(39), different efflux pump genes and amino acid substitutions within GyrA (Ser81Leu) and ParC (Ser84Leu; Glu88Ala) were detected among the isolates. The study genomes were closely related to four strains belonging to ST119. The isolates showed biofilm production, serum resistance, growth under iron limiting condition, surface-associated motility and adherence to host cell. Isolates induced cytokine production in the host cell and showed mice mortality. DISCUSSION AND CONCLUSIONS: This study is the first report of the presence of blaNDM-1 in A. pittii from India along with another carbapenemase blaOXA-58. Emergence of highly virulent, multidrug-resistant A. pittii with attributes similar to A. baumannii calls for surveillance to identify the novel strains and their pathogenic and resistance potential.

Genomics to detect transmission of livestock-associated methicillin-resistant Staphylococcus aureus from UK pigs in abattoirs during slaughter.

BACKGROUND: Livestock-associated MRSA (LA-MRSA) transmission/cross-contamination can occur at abattoir through colonized pigs, increasing occupational hazards and health concerns for workers. To assess this risk we used genomics to identify LA-MRSA lineages present in batches of pigs sent to slaughter and distribution of clones. METHODS: WGS was performed on 85 LA-MRSA previously isolated from six abattoirs from 105 batches of pigs sent from 100 UK farms. spa typing and MLST were performed on all isolates. A mashtree tree was constructed to compare genomes of the LA-MRSA with 1281 global isolates from livestock and humans. A phylogenetic tree and pairwise SNP distance matrices were built from whole genomes of 109 isolates closest to those from abattoirs to compare evolutionary relationships and identify clones. RESULTS: All abattoir isolates belonged to CC398 and were mainly of spa type t011, although other spa types were present. Phylogenetic analysis confirmed the abattoir isolates were most closely related to each other and to pig LA-MRSA from across Europe, indicating a common evolutionary origin with related lineages colonizing UK pigs.Comparison of genomes using SNPs suggested between one and four clones were transferring between pigs from different batches. Transmission likely occurred on farm premises, during transportation, and/or within abattoirs through contact with contaminated surfaces in lairage or post-stunning. CONCLUSIONS: Genomics forensically identified related isolates/clones circulating in pigs at slaughter, showing contamination occurs often. Results suggest that further genomic tracking will identify hotspots, and improvements in measures such as biosecurity and disinfection will help reduce risk for workers.

Recent dynamics in Neisseria gonorrhoeae genomic epidemiology in Brazil: antimicrobial resistance and genomic lineages in 2017-20 compared to 2015-16.

OBJECTIVES: Regular quality-assured WGS with antimicrobial resistance (AMR) and epidemiological data of patients is imperative to elucidate the shifting gonorrhoea epidemiology, nationally and internationally. We describe the dynamics of the gonococcal population in 11 cities in Brazil between 2017 and 2020 and elucidate emerging and disappearing gonococcal lineages associated with AMR, compare to Brazilian WGS and AMR data from 2015 to 2016, and explain recent changes in gonococcal AMR and gonorrhoea epidemiology. METHODS: WGS was performed using Illumina NextSeq 550 and genomes of 623 gonococcal isolates were used for downstream analysis. Molecular typing and AMR determinants were obtained and links between genomic lineages and AMR (determined by agar dilution/Etest) examined. RESULTS: Azithromycin resistance (15.6%, 97/623) had substantially increased and was mainly explained by clonal expansions of strains with 23S rRNA C2611T (mostly NG-STAR CC124) and mtr mosaics (mostly NG-STAR CC63, MLST ST9363). Resistance to ceftriaxone and cefixime remained at the same levels as in 2015-16, i.e. at 0% and 0.2% (1/623), respectively. Regarding novel gonorrhoea treatments, no known zoliflodacin-resistance gyrB mutations or gepotidacin-resistance gyrA mutations were found. Genomic lineages and sublineages showed a phylogenomic shift from sublineage A5 to sublineages A1-A4, while isolates within lineage B remained diverse in Brazil. CONCLUSIONS: Azithromycin resistance, mainly caused by 23S rRNA C2611T and mtrD mosaics/semi-mosaics, had substantially increased in Brazil. This mostly low-level azithromycin resistance may threaten the recommended ceftriaxone-azithromycin therapy, but the lack of ceftriaxone resistance is encouraging. Enhanced gonococcal AMR surveillance, including WGS, is imperative in Brazil and other Latin American and Caribbean countries.

Prolonged carriage of OXA-244-carbapenemase-producing Escherichia coli complicates epidemiological investigations.

The rapid increase of OXA-244-producing Escherichia coli, predominantly driven by genetically clustered isolates of sequence type (ST)38, has been observed in at least nine European countries, including Germany. However, the reasons for the spread of OXA-244-producing E. coli remain unclear. Here, we aim to evaluate the possibility of prolonged carriage. We identified a total of six different patients with repeated detection of OXA-244-producing E. coli isolates, which were subjected to both short and long-read whole-genome sequencing (WGS). Besides allelic differences using core genome multilocus sequence typing (cgMLST) analyses, we obtained numbers of single-nucleotide polymorphisms (SNPs) to calculate individual base-pair substitution (BPS) rates. To assess possible re-exposure and risk factors for prolonged carriage, case interviews were conducted. The time between detections ranged from eleven months to more than three years. Initial isolates originated in three+ out of six cases from clinical samples, whereas remaining samples were from screening, mostly in the inpatient setting. As expected, cgMLST analyses showed low numbers of allelic differences between isolates of each case ranging from 1 to 4, whereas numbers of SNPs were between 2 and 99 (mean = 36), thus clearly highlighting the discrepancy between these different bacterial typing approaches. For five out of six cases, observed BPS rates suggest that patients can be colonized with OXA-244-producing E. coli, including ST38 cluster isolates, for extensively long times. Thus, we may have previously missed the epidemiological link between cases because exposure to OXA-244-producing E. coli could have occurred in a time frame, which has not been evaluated in previous investigations. Our results may help to guide future epidemiological investigations as well as to support the interpretation of genetic diversity of OXA-244-producing E. coli, particularly among ST38 cluster isolates.

Survival after cryptococcosis in Germany: A retrospective multicenter cohort study of patients diagnosed between 2004 and 2021.

Cryptococcosis is the most prevalent fungal infection of the central nervous system worldwide. We performed a retrospective multicenter cohort study to gain insights into the epidemiology of cryptococcosis in Germany. We describe the use of diagnostic tests, clinical management and patient outcome. We included 64 patients with underlying HIV infection (55%) or other predispositions. Molecular typing by MLST documented 20 individual sequence types among 42 typed isolates. A fatal outcome was documented in 14% of patients in the first two months after diagnosis.

Prevalence and characterization of community-associated Staphylococcus aureus isolates from human mastitis in Beijing, China.

OBJECTIVES: Staphylococcus aureus (S. aureus) spreads worldwide and occurrence of mastitis caused by it holds significant implications for public health. We aim to reveal the molecular typing, antibiotic resistance and virulence gene profile of S. aureus causing mastitis through investigation. METHODS: A total of 200 isolates of S. aureus were collected from outpatients infected with mastitis in a hospital in Beijing from 2020.7 to 2021.7. The molecular characteristics were analyzed by MLST and spa typing, virulence genes were screened by PCR, antibiotic susceptible test was performed by VITEK® 2 Compact system and phylogenetic analysis was performed by MEGA11 and iTOL. RESULTS: Nineteen sequence types (STs) belonging to 9 clone complexes (CCs) were identified. ST22 was the most dominant clone (77.0%, 154/200). MRSA accounted for 19.0% (38/200) and 89.5% (34/38) of MRSA isolates belonged to CC22 and CC59. The isolates had relatively low levels of antibiotic resistance, with the exception of ß-lactams and macrolides with resistance rates above 50.0%. The carrying rate of pvl in the ST22-MRSA strains were 84.2% and the detection rates of seb and pvl in the MRSA isolates were significantly higher than those in the MSSA isolates, while the hlg, fnbA and sdrD showed opposite results. Whole genome sequenced specimens of MRSA strains X4 and B5 show the same evolutionary origin as ST22 EMRSA-15 (HE681097), which is popular in Europe. CONCLUSIONS: The method based on molecular epidemiology is an important tool for tracking the spread of S. aureus infections. We need to be alert to the major MRSA clones CC22 and CC59 in the region and be vigilant to the possible pandemic and spread of ST22 EMRSA-15.