Early identification of the nosocomial spread of vancomycin-resistant Enterococcus faecium by Fourier-transform infrared spectroscopy and performance comparison with PFGE and WGS.

Early detection of disseminating vancomycin-resistant Enterococcus faecium (VREfm) in ICU wards is crucial for outbreak identification and the implementation of prompt infection control measures. Genotypic methods like pulsed-field gel electrophoresis (PFGE) and whole-genome sequencing (WGS) are costly and time-consuming, hindering rapid response due to batch dependency. Fourier-transform infrared spectroscopy (FT-IR) offers the potential for real-time outbreak detection and reliable strain typing. We utilized FT-IR to identify clonal VREfm dissemination and compared its performance to PFGE and WGS. Between February through October 2023, an unusually high number of VREfm were recovered at a tertiary hospital in Barcelona. Isolates were examined for antimicrobial susceptibility, carriage of vanA/vanB genes and clonality was also studied using FT-IR, PFGE, and WGS. Routine FT-IR inspections revealed recurring VREfm clustering during the outbreak's initial weeks. In total, 104 isolates were recovered from 75 patients and from multiple wards. However, only one isolate was recovered from an environmental sample, suggesting the absence of environmental reservoirs. An ST80 vancomycin-resistant (vanA) E. faecium strain was the main strain responsible for the outbreak, although a few additional VREfm strains were also identified, all belonging to CC17. PFGE and cgMLST (WGS) yielded identical clustering results to FT-IR, and WGS confirmed vanA/vanB gene carriage in all VREfm isolates. Infection control measures led to a rapid decline in VREfm isolates, with no isolates detected in November. FT-IR spectroscopy offers rapid turnaround times, sensitivity, and reproducibility, comparable to standard typing methods. It proved as an effective tool for monitoring VREfm dissemination and early outbreak detection.

Adult T-cell leukemia/lymphoma in HTLV-1 non-endemic regions.

BACKGROUND: HTLV-1 infection is a neglected disease, despite producing neurological and lymphoproliferative severe illnesses and affect over 10 million people worldwide. Roughly 5% of HTLV-1 carriers develop Adult T-cell leukemia/lymphoma (ATLL), one of the most aggressive hematological malignancies. METHODS: A national HTLV-1 register exists since 1989 in Spain, a non-endemic country with a large migrant flow from Latin America and Equatorial Africa, where HTLV-1 is endemic. The main features of all patients diagnosed with ATLL in Spain up to date are reported. RESULTS: A total of 451 cases of HTLV-1 infection had been reported in Spain until the end of year 2022. ATLL had been diagnosed in 35 (7.8%). The current average incidence of ATLL in Spain is of two cases per year. Women represent 57% of ATLL patients. Mean age at diagnosis was 47 years-old. Roughly 57% were Latin Americans and 26% Africans. At diagnosis, the majority presented with acute or lymphoma clinical forms. Survival was shorter than one year in most of them. Mean HTLV-1 proviral load was significantly greater in ATLL patients than in asymptomatic HTLV-1 carriers (2,305 vs 104 copies/104 PBMC). HTLV-1 subtyping in 6 ATLL patients found the 1a transcontinental variant (n = 4) and the Japanese variant (n = 2). All ATLL patients were negative for HIV-1, did not develop HTLV-1-associated myelopathy and were not transplant recipients. CONCLUSION: The rate of ATLL is very low in Spain and mostly associated to migrants from HTLV-1 endemic regions. Given the poor clinical outcome of ATLL, HTLV-1 testing should be performed at least once in all migrants coming from HTLV-1 endemic countries and in natives who have lived in or had sex partners from such regions.

The slowdown of new infections by human retroviruses has reached a plateau in Spain.

The 2022 annual meeting of the HTLV & HIV-2 Spanish Network was held in Madrid on December 14. We summarize here the main information presented and discussed at the workshop and review time trends for human retroviral infections in Spain. As transmissible agents, infections by human retroviruses are of obligatory declaration. Until the end of 2022, the Spanish national registry had recorded 451 cases of HTLV-1, 821 of HTLV-2, and 416 of HIV-2. For HIV-1, estimates are of 150 000 people currently living with HIV-1 and 60 000 cumulative deaths due to AIDS. During year 2022, new diagnoses in Spain were of 22 for HTLV-1, 6 for HTLV-2, and 7 for HIV-2. The last updated figures for HIV-1 are from 2021 and counted 2786 new diagnoses. The slowdown in yearly infections for HIV-1 in Spain points out that new strategies are needed to achieve the United Nations 95-95-95 targets by 2025. For the remaining neglected human retroviral infections, their control might be pushed throughout four interventions: (1) expanding testing; (2) improving education and interventions aimed to reduce risk behaviors; (3) facilitating access to antiretrovirals as treatment and prevention, including further development of long-acting formulations; and (4) increasing vaccine research efforts. Spain is a 47 million population country in South Europe with strong migration flows from HTLV-1 endemic regions in Latin America and Sub-Saharan Africa. At this time universal HTLV screening has been implemented only in the transplantation setting, following the report of 5 cases of HTLV-associated myelopathy shortly after transplantation of organs from HTLV-1 positive donors. There are four target populations for expanding testing and unveiling asymptomatic carriers responsible for silent HTLV-1 transmissions: (1) migrants; (2) individuals with sexually transmitted infections; (3) pregnant women; and (4) blood donors.

Susceptibility to hepatitis B virus infection in adults living in Spain.

BACKGROUND: A protective hepatitis B virus (HBV) vaccine has been available for four decades. Universal HBV vaccination of infants is recommended by the WHO since the 1990s. Furthermore, HBV immunization is advised for all adults with high-risk behaviours and no seroprotection. However, HBV vaccine coverage remains globally suboptimal. The advent of new more efficacious trivalent HBV vaccines has renewed the interest in HBV vaccination. At present, the extent of current HBV susceptibility in adults remains unknown in Spain. METHODS: HBV serological markers were assessed on a large and representative sample of adults in Spain, including blood donors and individuals belonging to high-risk groups. Serum HBsAg, anti-HBc and anti-HBs were tested in specimens collected during the last couple of years. RESULTS: From 13 859 consecutive adults tested at seven cities across the Spanish geography, overall 166 (1.2%) had positive HBsAg. Past HBV infection was recognized in 14% and prior vaccine immunization in 24%. Unexpectedly, 37% of blood donors and 63% of persons belonging to high-risk groups had no serum HBV markers and therefore were potentially HBV susceptible. CONCLUSION: Roughly 60% of adults living in Spain seem to be HBV susceptible. Waning immunity might be more common than expected. Hence, HBV serological testing should be performed at least once in all adults regardless of risk exposures. HBV vaccine full courses or boosters should be administered to all adults lacking serological evidence of HBV protection.

Host-associated variability of the cdtABC operon, coding for the cytolethal distending toxin, in Campylobacter jejuni.

Campylobacter, a major cause of food-borne gastroenteritis worldwide, colonize the gastrointestinal tract of a wide range of animals, being birds the main reservoir. The mechanisms involved in the interaction of Campylobacter with the different hosts are poorly understood. The cytolethal distending toxin, encoded in the cdtABC operon, is considered a pivotal virulence factor during human infection. Differences in the prevalence of cdtABC genes in Campylobacter isolates from three distinct origins (wild birds, broiler chickens and humans) prompted us to further characterize their allelic variability. The sequence of cdtABC is highly conserved among broiler and human isolates. A high diversity of cdtABC alleles was found among wild bird isolates, including several alleles that do not produce any functional CDT. These results suggest that specific variants of the cdtABC operon might define the host range of specific Campylobacter jejuni isolates. Moreover, our data indicate that PCR methodology is inaccurate to characterize the prevalence of the cdt genes, since negative PCR detection can be the result of divergences in the sequence used for primer design rather than indicating the absence of a specific gene.

Late presentation of human T-lymphotropic virus type 1 infection in Spain reflects suboptimal testing strategies.

OBJECTIVES: Although only 10% of persons infected with human T-lymphotropic virus type 1 (HTLV-1) may develop virus-associated illnesses over their lifetime, missing the earlier diagnosis of asymptomatic carriers frequently leads to late presentation. METHODS: A nationwide HTLV-1 register was created in Spain in 1989. We examined the main demographics and clinical features at the time of the first diagnosis for more than three decades. RESULTS: A total of 428 individuals infected with HTLV-1 had been reported in Spain until the end of 2021. Up to 96 (22%) individuals presented clinically with HTLV-1-associated conditions, including subacute myelopathy (57%), T-cell lymphoma (34%), or Strongyloides stercoralis infestation (8%). Since 2008, HTLV-1 diagnosis has been made at blood banks (44%) or clinics (56%). Native Spaniards and Sub-Saharan Africans are overrepresented among patients presenting with HTLV-1-associated illnesses suggesting that poor epidemiological and/or clinical suspicion, which led to the late presentation are more frequent in them than carriers from Latin America (LATAM) (31.7% vs 20.4%, respectively; P = 0.015). CONCLUSION: HTLV-1 infection in Spain is frequently diagnosed in patients presenting with characteristic illnesses. Although screening in blood banks mostly identifies asymptomatic carriers from LATAM, a disproportionately high number of Spaniards and Africans are diagnosed too late at the time of clinical manifestations. Expanding testing to all pregnant women and clinics for sexually transmitted infections could help to unveil HTLV-1 asymptomatic carriers.

Viral culture and immunofluorescence for the detection of SARS-CoV-2 infectivity in RT-PCR positive respiratory samples.

BACKGROUND: Knowing how long SARS-CoV-2-positive individuals can remain infective is crucial for the design of infection prevention and control strategies. Viral culture is the gold standard for detecting an active-replicative virus and evaluating its infectious potential. OBJECTIVE: To assess the correlation of SARS-CoV-2 infectivity with the number of days from symptom onset and the Ct value, using culture as a reference method. Also, to describe a detailed protocol for SARS-CoV-2 culture and immunofluorescence confirmation based on our experience with other respiratory viruses. STUDY DESIGN: 100 consecutive respiratory samples positive for SARS-CoV-2 by RT-PCR from different subjects were inoculated into VERO E6 cells. RESULTS: Viral isolation was successful in 58% of samples. The median number of days from symptom onset for culture-positive samples was 2, and 15 for culture-negative samples. Six positive cultures were obtained in patients ≥14 days after symptom onset, all of whom were immunocompromised or with severe COVID-19. The mean Ct value was 12.64 units higher in culture-negative than in culture-positive samples. The probability of successfully isolating SARS-CoV-2 in samples with a Ct value <22 was 100%, decreasing to 3.1% when >27. CONCLUSIONS: Our findings show a significant positive correlation between the probability of isolating SARS-CoV-2 in culture, fewer days of symptoms and a lower RT-PCR Ct value. SARS-CoV-2 infectivity lasts no more than 14 days from symptom onset in immunocompetent individuals. In contrast, in immunocompromised patients or those with severe COVID-19 infectivity may remain after 14 days. Ct value <22 always indicates infectivity.

A New Variant of the aadE-sat4-aphA-3 Gene Cluster Found in a Conjugative Plasmid from a MDR Campylobacter jejuni Isolate.

Campylobacter jejuni is a foodborne pathogen causing bacterial gastroenteritis, with the highest incidence reported in Europe. The prevalence of antibiotic resistance in C. jejuni, as well as in many other bacterial pathogens, has increased over the last few years. In this report, we describe the presence of a plasmid in a multi-drug-resistant C. jejuni strain isolated from a gastroenteritis patient. Mating experiments demonstrated the transference of this genetic element (pCjH01) among C. jejuni by plasmid conjugation. The pCjH01 plasmid was sequenced and assembled, revealing high similarity (97% identity) with pTet, a described tetracycline resistance encoding plasmid. pCjH01 (47.7 kb) is a mosaic plasmid composed of a pTet backbone that has acquired two discrete DNA regions. Remarkably, one of the acquired sequences carried an undescribed variant of the aadE-sat4-aphA-3 gene cluster, providing resistance to at least kanamycin and gentamycin. Aside from the antibiotic resistance genes, the cluster also carries genes coding for putative regulators, such as a sigma factor of the RNA polymerase and an antisigma factor. Homology searches suggest that Campylobacter exchanges genetic material with distant G-positive bacterial genera.

Viral Culture Confirmed SARS-CoV-2 Subgenomic RNA Value as a Good Surrogate Marker of Infectivity.

Determining SARS-CoV-2 viral infectivity is crucial for patient clinical assessment and isolation decisions. We assessed subgenomic RNA (sgRNA) as a surrogate marker of SARS-CoV-2 infectivity in SARS-CoV-2-positive reverse transcription PCR (RT-PCR) respiratory samples (n = 105) in comparison with viral culture as the reference standard for virus replication. sgRNA and viral isolation results were concordant in 99/105 cases (94%), indicating highly significant agreement between the two techniques (Cohen's kappa coefficient 0.88, 95% confidence interval [CI] 0.78 to 0.97, P < 0.001). sgRNA RT-PCR showed a sensitivity of 97% and a positive predictive value of 94% to detect replication-competent virus, further supporting sgRNA as a surrogate marker of SARS-CoV-2 infectivity. sgRNA RT-PCR is an accurate, rapid, and affordable technique that can overcome culture and cycle threshold (CT) value limitations and be routinely implemented in hospital laboratories to detect viral infectivity, which is essential for optimizing patient monitoring, the efficacy of treatments/vaccines, and work reincorporation policies, as well as for safely shortening isolation precautions.

Taxonomic Identification of Different Species of the Genus Aeromonas by Whole-Genome Sequencing and Use of Their Species-Specific ß-Lactamases as Phylogenetic Markers.

Some Aeromonas species, potentially pathogenic for humans, are known to express up to three different classes of chromosomal ß-lactamases, which may become hyperproduced and cause treatment failure. The aim of this study was to assess the utility of these species-specific ß-lactamase genes as phylogenetic markers using whole-genome sequencing data. Core-genome alignments were generated for 36 Aeromonas genomes from seven different species and scanned for antimicrobial resistance genes. Core-genome alignment confirmed the MALDI-TOF identification of most of the isolates and re-identified an A. hydrophila isolate as A. dhakensis. Three (B, C and D) of the four Ambler classes of ß-lactamase genes were found in A. sobria, A. allosacharophila, A. hydrophila and A. dhakensis (blaCphA, blaAmpC and blaOXA). A. veronii only showed class-B- and class-D-like matches (blaCphA and blaOXA), whereas those for A. media, A. rivipollensis and A. caviae were class C and D (blaCMY, blaMOX and blaOXA427). The phylogenetic tree derived from concatenated sequences of ß-lactamase genes successfully clustered each species. Some isolates also had resistance to sulfonamides, quinolones and aminoglycosides. Whole-genome sequencing proved to be a useful method to identify Aeromonas at the species level, which led to the unexpected identification of A. dhakensis and A.rivipollensis and revealed the resistome of each isolate.

Whole-genome analysis to describe a human adenovirus D8 conjunctivitis outbreak in a tertiary hospital.

Conjunctivitis is a frequent ocular disorder caused by human adenoviruses (HAdVs). Only a few of the 45 HAdV-D species are associated with epidemic keratoconjunctivitis, including HAdV-D8. Nosocomial outbreaks due to HAdV-D8 have been rarely described, because keratoconjunctivitis cases are clinically diagnosed and treated without having to characterize the causative agent. Moreover, molecular typing is tedious when using classical techniques. In this study, a hospital outbreak of conjunctivitis caused by HAdV-D8 was characterized using the recently developed whole-genome sequencing (WGS) method. Of the 363 patients attending the Ophthalmology Department between July 13 and August 13, 2018, 36 may have acquired intrahospital conjunctivitis. Also, 11 of 22 samples sent to the Virology section were selected for WGS analysis. The WGS results revealed that 10 out of 11 HAdV-D8 strains were closely related. The remaining strain (Case 28) was more similar to a strain from an outbreak in Germany obtained from a public sequence database. WGS results showed that outbreak HAdV-D8 strains had a minimum percentage of identity of 94.3%. WGS is useful in a clinical setting, because it avoids carrying out viral culture or specific polymerase chain reaction sequencing. The public availability of sequence reads makes it easier to compare clusters in circulation. In conclusion, WGS can play an important role in standard routines to describe viral outbreaks.

Genomic analysis of 40 prophages located in the genomes of 16 carbapenemase-producing clinical strains of Klebsiella pneumoniae.

Klebsiella pneumoniae is the clinically most important species within the genus Klebsiella and, as a result of the continuous emergence of multi-drug resistant (MDR) strains, the cause of severe nosocomial infections. The decline in the effectiveness of antibiotic treatments for infections caused by MDR bacteria has generated particular interest in the study of bacteriophages. In this study, we characterized a total of 40 temperate bacteriophages (prophages) with a genome range of 11.454-84.199 kb, predicted from 16 carbapenemase-producing clinical strains of K. pneumoniae belonging to different sequence types, previously identified by multilocus sequence typing. These prophages were grouped into the three families in the order Caudovirales (27 prophages belonging to the family Myoviridae, 10 prophages belonging to the family Siphoviridae and 3 prophages belonging to the family Podoviridae). Genomic comparison of the 40 prophage genomes led to the identification of four prophages isolated from different strains and of genome sizes of around 33.3, 36.1, 39.6 and 42.6 kb. These prophages showed sequence similarities (query cover >90 %, identity >99.9 %) with international Microbe Versus Phage (MVP) (http://mvp.medgenius.info/home) clusters 4762, 4901, 3499 and 4280, respectively. Phylogenetic analysis revealed the evolutionary proximity among the members of the four groups of the most frequently identified prophages in the bacterial genomes studied (33.3, 36.1, 39.6 and 42.6 kb), with bootstrap values of 100 %. This allowed the prophages to be classified into three clusters: A, B and C. Interestingly, these temperate bacteriophages did not infect the highest number of strains as indicated by a host-range assay, these results could be explained by the development of superinfection exclusion mechanisms. In addition, bioinformatic analysis of the 40 identified prophages revealed the presence of 2363 proteins. In total, 59.7 % of the proteins identified had a predicted function, mainly involving viral structure, transcription, replication and regulation (lysogenic/lysis). Interestingly, some proteins had putative functions associated with bacterial virulence (toxin expression and efflux pump regulators), phage defence profiles such as toxin-antitoxin modules, an anti-CRISPR/Cas9 protein, TerB protein (from terZABCDE operon) and methyltransferase proteins.

Unravelling the consequences of the bacteriophages in human samples.

Bacteriophages are abundant in human biomes and therefore in human clinical samples. Although this is usually not considered, they might interfere with the recovery of bacterial pathogens at two levels: 1) by propagating in the enrichment cultures used to isolate the infectious agent, causing the lysis of the bacterial host and 2) by the detection of bacterial genes inside the phage capsids that mislead the presence of the bacterial pathogen. To unravel these interferences, human samples (n = 271) were analyzed and infectious phages were observed in 11% of blood culture, 28% of serum, 45% of ascitic fluid, 14% of cerebrospinal fluid and 23% of urine samples. The genetic content of phage particles from a pool of urine and ascitic fluid samples corresponded to bacteriophages infecting different bacterial genera. In addition, many bacterial genes packaged in the phage capsids, including antibiotic resistance genes and 16S rRNA genes, were detected in the viromes. Phage interference can be minimized applying a simple procedure that reduced the content of phages up to 3 logs while maintaining the bacterial load. This method reduced the detection of phage genes avoiding the interference with molecular detection of bacteria and reduced the phage propagation in the cultures, enhancing the recovery of bacteria up to 6 logs.

Pathogenesis of Staphylococcus epidermidis in prosthetic joint infections: Can identification of virulence genes differentiate between infecting and commensal strains?

BACKGROUND: Staphylococcus epidermidis is a commensal of human skin flora and a frequent causative microorganism in prosthetic joint infections (PJIs). To date, no single marker has been identified to distinguish infecting strains from commensal S. epidermidis populations. AIM: We aimed to find possible genetic markers to distinguish between the two populations. METHODS: We analyzed 50 S. epidermidis strains from patients with PJIs, 50 from skin of healthy individuals (commensal strains) and 17 from the surgical field of patients undergoing primary arthroplasty. In these three groups we studied the antimicrobial susceptibility profile, sequence type, biofilm formation, and virulence factors. Strains from the surgical field have not been compared previously with strains from the other two groups. FINDINGS: S. epidermidis strains from PJI patients were significantly more antibiotic resistant than commensal strains and surgical field strains. A wide variety of sequences types was found in commensal and surgical field strains. The predominant sequence type was ST2 and it was only present in PJI strains (44%). Differences in biofilm production did not differ between populations. Virulence genes sdrF and bhp, the complete ica operon, and the insertion sequence IS256 were significantly predominant in PJI strains. In contrast, embp and hld genes and the mobile element ACME were more prevalent in commensal strains. Surgical field strains could be a valid control group to discriminate between infecting and commensal strains. CONCLUSION: A combination of characteristic features can differentiate between infecting and commensal S. epidermidis strains in PJI, while a single marker cannot.

In vitro and in vivo efficacy of combinations of colistin and different endolysins against clinical strains of multi-drug resistant pathogens.

The emergence of multidrug resistant (MDR) pathogenic bacteria is jeopardizing the value of antimicrobials, which had previously changed the course of medical science. In this study, we identified endolysins ElyA1 and ElyA2 (GH108-PG3 family), present in the genome of bacteriophages Ab1051Φ and Ab1052Φ, respectively. The muralytic activity of these endolysins against MDR clinical isolates (Acinetobacter baumannii, Pseudomonas aeruginosa and Klebsiella pneumoniae) was tested using the turbidity reduction assay. Minimal inhibitory concentrations (MICs) of endolysin, colistin and a combination of endolysin and colistin were determined, and the antimicrobial activity of each treatment was confirmed by time kill curves. Endolysin ElyA1 displayed activity against all 25 strains of A. baumannii and P. aeruginosa tested and against 13 out of 17 strains of K. pneumoniae. Endolysin ElyA2 did not display any such activity. The combined antimicrobial activity of colistin and ElyA1 yielded a reduction in the colistin MIC for all strains studied, except K. pneumoniae. These results were confirmed in vivo in G. mellonella survival assays and in murine skin and lung infection models. In conclusion, combining colistin (1/4 MIC) with the new endolysin ElyA1 (350 µg) enhanced the bactericidal activity of colistin in both in vitro and in vivo studies. This will potentially enable reduction of the dose of colistin used in clinical practice.

Comparison of Commensal and Clinical Isolates for Diversity of Plasmids in Escherichia coli and Klebsiella pneumoniae.

In this study, the plasmid content of clinical and commensal strains was analyzed and compared. The replicon profile was similar in both populations, except for L, M, A/C, and N (detected only in clinical strains) and HI1 (only in commensal strains). Although I1 and F were the most frequent replicons, only IncI1, sequence type 12 (ST12) was associated with blaCMY-2 in both populations. In contrast, the widespread resistant IncF plasmids were not linked to a single epidemic plasmid.

Differential Distribution of the wlaN and cgtB Genes, Associated with Guillain-Barré Syndrome, in Campylobacter jejuni Isolates from Humans, Broiler Chickens, and Wild Birds.

Campylobacter jejuni causes campylobacteriosis, a bacterial gastroenteritis with high incidence worldwide. Moreover, C. jejuni infection can trigger the polyneuropathic disorder denominated Guillain-Barré syndrome (GBS). The C. jejuni strains that can elicit GBS carry either wlaN or cgtB, coding both genes for a ß-1,3-galactosyltransferase enzyme that is required for the production of sialylated lipooligosaccharide (LOSSIAL). We described a differential prevalence of the genes wlaN and cgtB in C. jejuni isolates from three different ecological niches: humans, broiler chickens, and wild birds. The distribution of both genes, which is similar between broiler chicken and human isolates and distinct when compared to the wild bird isolates, suggests a host-dependent distribution. Moreover, the prevalence of the wlaN and cgtB genes seems to be restricted to some clonal complexes. Gene sequencing identified the presence of new variants of the G- homopolymeric tract within the wlaN gene. Furthermore, we detected two variants of a G rich region within the cgtB gene, suggesting that, similarly to wlaN, the G-tract in the cgtB gene mediates the phase variation control of cgtB expression. Caco-2 cell invasion assays indicate that there is no evident correlation between the production of LOSSIAL and the ability to invade eukaryotic cells.

Faecal phageome of healthy individuals: presence of antibiotic resistance genes and variations caused by ciprofloxacin treatment.

OBJECTIVES: Antimicrobial resistance genes (ARGs) can be transferred by means of mobile genetic elements, which play a critical role in the dissemination of resistance in the bacterial community. ARG transmission within mobile genetic elements has been reported in plasmids and transposons but less frequently in bacteriophages. Here, the bacteriophage fraction of seven human faecal samples was purified and deep-sequenced to detect the presence of ARGs in the phage particles. METHODS: Seven faecal samples (five from healthy individuals and two from a patient before and after receiving ciprofloxacin treatment) were used to extract phage DNA, which was purified and then sequenced in a MiSeq (Illumina). Generated reads were checked for quality and assembled, and then the generated contigs analysed with Kraken, PHASTER, VirSorter and Prokka. Some genes were also validated by quantitative PCR. RESULTS: Analysis of the purified phage DNA by Kraken identified from 4 to 266 viruses in the samples. The viral fraction corresponded mainly to the order Caudovirales, including phages from the Siphoviridae and Myoviridae families. Bacterial genes associated with antimicrobial resistance were detected in the viral DNA, as confirmed by quantitative PCR. Higher densities of ARG-carrying phage particles were observed in the post- versus pre-ciprofloxacin treatment sample. CONCLUSIONS: The finding of ARGs in phage particles supports the description of phages as mobile elements contributing to the dissemination of bacterial antibiotic resistance and suggests ciprofloxacin treatment may play a role in the release of ARG-carrying particles, thereby increasing resistance.

Molecular characterization of OXA-48 carbapenemase-producing Klebsiella pneumoniae strains after a carbapenem resistance increase in Catalonia.

INTRODUCTION: To characterize OXA-48 carbapenemase-producing Klebsiella pneumoniae strains isolated after an increase in carbapenem resistance in Catalonia. METHODOLOGY: K. pneumoniae identification, antimicrobial susceptibility studies, the Modified Hodge Test method, amplification of antimicrobial resistance genes (against ß-lactamases, quinolones and aminoglycosides), molecular typing (by PFGE and MLST), conjugation assays, plasmid characterization (PBRT-PCR and Southern blot), a description of mobile genetic elements and statistical analysis were done. RESULTS: OXA-48 was the only carbapenemase detected, with a prevalence of 1.9%. The blaOXA-48 gene was located in an IncL conjugative plasmid of 62kb and integrated into the transposons Tn1999.2 (91.7%) or Tn1999.1. Five PFGE profiles (A to E) were found, which exactly matched the MLST: ST101, ST17, ST1233, ST14 and ST405, respectively. ST1233 is described here for the first time. K. pneumoniae OXA-48-producing strains were also CTX-M-15 carriers, some producing OXA-1 and TEM-1 penicillinases. The acquired qnrB66 and qnrB1 and aac(3')-IIa, aac(6')-Ib genes were also identified. CONCLUSION: The K. pneumoniae ST405 clone has played an important role in the growing prevalence of OXA-48 in Catalonia. All clones described preserved the blaOXA-48 genetic environment and mobile genetic elements (Tn1999). Notably, the three strains with minor sequence types in this study are not multiresistant strains. These strains are expanding in elderly patients (average age of 76 years) with serious underlying diseases, mainly women (61.2%).

Core/Whole Genome Multilocus Sequence Typing and Core Genome SNP-Based Typing of OXA-48-Producing Klebsiella pneumoniae Clinical Isolates From Spain.

Whole-genome sequencing (WGS)-based typing methods have emerged as promising and highly discriminative epidemiological tools. In this study, we combined gene-by-gene allele calling and core genome single nucleotide polymorphism (cgSNP) approaches to investigate the genetic relatedness of a well-characterized collection of OXA-48-producing Klebsiella pneumoniae isolates. We included isolates from the predominant sequence type ST405 (n = 31) OXA-48-producing K. pneumoniae clone and isolates from ST101 (n = 3), ST14 (n = 1), ST17 (n = 1), and ST1233 (n = 1), obtained from eight Catalan hospitals. Core-genome multilocus sequence typing (cgMLST) schemes from Institut Pasteur's BIGSdb-Kp (634 genes) and SeqSphere+ (2,365 genes), and a SeqSphere+ whole-genome MLST (wgMLST) scheme (4,891 genes) were used. Allele differences or allelic mismatches and the genetic distance, as the proportion of allele differences, were used to interpret the results from a gene-by-gene approach, whereas the number of SNPs was used for the cgSNP analysis. We observed between 0-10 and 0-14 allele differences among the predominant ST405 using cgMLST and wgMLST from SeqSphere+, respectively, and <2 allelic mismatches when using Institut Pasteur's BIGSdb-Kp cgMLST scheme. For ST101, we observed 14 and 54 allele differences when using cgMLST and wgMLST SeqSphere+, respectively, and 2-5 allelic mismatches for BIGSdb-Kp cgMLST. A low genetic distance (<0.0035, a previously established threshold for epidemiological link) was generally in concordance with a low number of allele differences (<8) when using the SeqSphere+ cgMLST scheme. The cgSNP analysis showed 6-29 SNPs in isolates with identical allelic SeqSphere+ cgMLST profiles and 16-61 cgSNPs among ST405 isolates. Furthermore, comparison of WGS-based typing results with previously obtained MLST and pulsed-field gel electrophoresis (PFGE) data showed some differences, demonstrating the different molecular principles underlying these techniques. In conclusion, the use of the different WGS-based typing methods that were used to elucidate the genetic relatedness of clonal OXA-48-producing K. pneumoniae all led to the same conclusions. Furthermore, threshold parameters in WGS-based typing methods should be applied with caution and should be used in combination with clinical epidemiological data and population and species characteristics.