Interhospital Spread of blaVIM-1- and blaCTX-M-15-Producing K. pneumoniae ST15 on an IncR Plasmid in Southern Spain.

In 2014-2015, the main CTX-M-15- and OXA-48-producing clone in our region was ST15. Recently, K. pneumoniae ST15 isolates co-producing VIM-1 and CTX-M-15 were detected in several hospitals. The aim was to study the emergence and acquisition of this carbapenemase. Between 2017 and 2019, four hospitals submitted twenty-nine VIM-1- and CTX-M-15-producing K. pneumoniae ST15 isolates to our laboratory. Seven representatives of each XbaI PFGE pulsotype were sequenced using short- and long-read technologies. RAST, CGE databases, and Pathogenwatch were used for resistance determinants and capsule-type analysis. Plasmid comparison was performed with Easyfig2.1. Phylogenetic analysis included other contemporary ST15 isolates from Spain. The 29 isolates were clustered into seven different pulsotypes. The selected genomes, from three hospitals in two different provinces, were clustered together (fewer than 35 alleles) and differed by more than 100 alleles from other ST15 isolates obtained in the region. These seven isolates harbored one IncR plasmid (200-220 kb) with a common backbone and four regions flanked by IS26: one contained blaVIM-1, another contained blaCTX-M-15, the third contained blaOXA-1, and the fourth harbored heavy-metal-tolerance genes. The two initial plasmids, from two different centers, were identical, and rearrangement of four regions was observed in the five subsequent plasmids. Our findings showed the first intercenter dissemination of IncR plasmids carrying blaVIM-1, blaCTX-M-15, and metal-tolerance genes mediated by a new lineage of K. pneumoniae ST15. Two different capture events of the blaVIM-1 gene or different IS26-mediated plasmid rearrangements from a common ancestor may explain plasmid variations.

Molecular characterization of a suspected IMP-type carbapenemase-producing Pseudomonas aeruginosa outbreak reveals two simultaneous outbreaks in a tertiary-care hospital.

OBJECTIVE: To describe IMP-type carbapenemase-producing Pseudomonas aeruginosa outbreaks at Galdakao University Hospital between March 2021 to December 2021. DESIGN: Outbreak report. SETTING: Galdakao University Hospital is a tertiary-care hospital in the Basque Country (northern Spain). PATIENTS: All patients with a positive IMP-type carbapenemase producing Pseudomonas aeruginosa (IMP-PA) culture were included in this study, both colonization and infection cases. METHODS: An outbreak investigation was conducted, in which molecular epidemiology analysis [pulsed-field gel electrophoresis and whole-genome sequencing (WGS)] and environmental screenings were performed. RESULTS: Between March and December 2021, 21 cases of IMP-PA were detected in Galdakao University Hospital: 18 infection cases and 3 colonization cases. In total, 4 different pulsotypes were detected belonging to 4 clones according to WGS: ST175 (n = 14), ST633 (n = 3), ST179 (n = 3), and ST348 (n = 1). IMP-13 was detected in most isolates belonging to the ST175 clone and in all ST179 and ST348 clones, whereas IMP-29 was detected in isolates belonging to the ST633 clone. Clinical isolates belonging to the ST175 clone were isolated mainly from patients admitted to the respiratory ward, and isolates belonging to the ST633 clone from patients admitted to the ICU. Two environmental isolates belonging to the ST175 clone were detected in the respiratory ward. CONCLUSIONS: Molecular and genomic epidemiology revealed that there had been 2 independent IMP-PA outbreaks, one of long duration in the respiratory ward and the other more limited in the ICU.

Prevalence of the fimbrial operon mrkABCD, mrkA expression, biofilm formation and effect of biocides on biofilm formation in carbapenemase-producing Klebsiella pneumoniae isolates belonging or not belonging to high-risk clones.

BACKGROUND: The role of mrkA adhesin expression, biofilm production, biofilm viability and biocides in the biofilm of carbapenemase-producing Klebsiella pneumoniae isolates was investigated. METHODS: Seventeen isolates representing different sequence types and carbapenemases were investigated. mrkA expression was determined by real-time reverse transcription polymerase chain reaction. Biofilm production (25°C and 37°C, with and without humidity) was determined by the crystal violet assay. The effect of isopropanol, povidone-iodine, sodium hypochlorite, chlorhexidine digluconate, benzalkonium chloride, ethanol and triclosan on biofilm was determined. The effect of povidone-iodine on biofilm biomass and thickness was also determined by confocal laser scanning microscopy. RESULTS: mrkA expression ranged from 28.2 to 1.3 [high or intermediate level; 64% of high-risk (HR) clones] and from 21.5 to 1.3 (50% of non-HR clones). At 25°C, biofilm formation was observed in 41% of isolates (absence of humidity) and 35% of isolates (presence of humidity), whereas at 37°C, biofilm formation was observed in 76% of isolates with and without humidity. At 25°C, biofilm producers were more frequently observed in HR clones (45% with humidity and 55% without humidity) than non-HR clones (17% with and without humidity). Biofilm viability from day 21 was higher at 25°C than 37°C. The greatest decrease in biofilm formation was observed with povidone-iodine (29% decrease), which also decreased biofilm thickness. CONCLUSIONS: Biofilm formation in carbapenemase-producing K. pneumoniae is related to mrkA expression. Biofilm formation is affected by temperature (37°C>25°C), whereas humidity has little effect. Biofilm viability is affected by temperature (25°C>37°C). At 25°C, HR clones are more frequently biofilm producers than non-HR clones. Povidone-iodine can decrease biofilm production and biofilm thickness.

In vitro activity of six biocides against carbapenemase-producing Klebsiella pneumoniae and presence of genes encoding efflux pumps / Actividad in vitro de seis biocidas frente a Klebsiella pneumoniae productora de carbapenemasa y presencia de genes codificantes de bombas de expulsión

Introduction: Acquisition of reduced susceptibility to biocides may contribute to the dissemination of high-risk (HR) clones of carbapenemase-producing Klebsiella pneumoniae (CP-Kp). The aim of this study was (a) to determinate the activity of biocides against CP-Kp, and (b) to analyse the relationship between biocide activity and the presence of efflux pumps. Methods: The minimal inhibitory concentrations (MICs) of 6 biocides (sodium hypochlorite, chlorhexidine digluconate, benzalkonium chloride, povidone-iodine, ethanol and triclosan) were determined in triplicate at 25°C and 37°C in Mueller-Hinton broth (MHB) and M9 minimum medium, against 17 CP-Kp isolates representing different clones (HR and no-HR), sequence-types (STs) and carbapenemases. Efflux pumps genes were detected by whole genome sequencing (MiSeq). Results: Median MICs were slightly higher at 37°C than at 25°C (p≤0.05), except for benzalkonium chloride, triclosan and ethanol. MIC medians were much higher in MHB than in M9, except for triclosan. No significant differences were observed in the median MICs, regarding the type of clone, ST or carbapenemase; cepA, acrAB, kpnEF and oqxAB genes were detected in all isolates, whereas qacE and qacA were not detected; smvAR, and qacΔE genes were detected in 94% and 47% of isolates, respectively. Conclusions: Triclosan, chlorhexidine digluconate, benzalkonium chloride and ethanol were the most active biocides. The activity of some biocides is affected by temperature and growth media, suggesting that standardised procedures for biocide susceptibility testing based on MIC determination are required. This activity, in terms of MICs, are not related to the type of clone, ST, carbapenemase or the presence of the efflux pump genes.(AU)

Introducción: La adquisición de sensibilidad reducida a los biocidas puede contribuir a la diseminación de clones de alto riesgo (HR) de Klebsiella pneumoniae productor de carbapenemasa (Kp-PC). El objetivo de este trabajo fue: (a) determinar la actividad de varios biocidas frente a Kp-PC, y (b) analizar la relación de dicha actividad con la presencia de genes codificantes de bombas de expulsión. Métodos: Las concentraciones mínimas inhibitorias (CMI) de 6 biocidas (hipoclorito de sodio, digluconato de clorhexidina, cloruro de benzalconio, povidona yodada, etanol y triclosán) se determinaron por triplicado a 25 y 37°C, tanto en caldo Mueller-Hinton (MHB) como en medio mínimo M9, frente a 17 aislados de Kp-PC representativos de diferentes clones (HR y no HR), secuenciotipos (ST) y carbapenemasas. Los genes de bombas de expulsión se detectaron mediante secuenciación masiva del genoma completo (MiSeq). Resultados: Las medianas de las CMI fueron ligeramente superiores a 37°C que a 25°C, excepto para cloruro de benzalconio, etanol y triclosán. Las medianas de las CMI fueron considerablemente superiores en MHB que en M9, excepto para triclosán; cepA, acrAB, kpnEF y oqxAB se detectaron en todos los aislados, mientras que qacE y qacA no se detectaron; smvAR y qacΔE se detectaron en el 94% y en el 47% de los aislados, respectivamente. Conclusiones: La actividad de algunos biocidas se afecta por la temperatura y el medio de crecimiento. Esta actividad, en términos de CMI, no se relaciona con el tipo de clon, ST, carbapenemasa, ni con la presencia de genes que codifican bombas de expulsión.(AU)

In vitro activity of six biocides against carbapenemase-producing Klebsiella pneumoniae and presence of genes encoding efflux pumps.

INTRODUCTION: Acquisition of reduced susceptibility to biocides may contribute to the dissemination of high-risk (HR) clones of carbapenemase-producing Klebsiella pneumoniae (CP-Kp). The aim of this study was (a) to determinate the activity of biocides against CP-Kp, and (b) to analyse the relationship between biocide activity and the presence of efflux pumps. METHODS: The minimal inhibitory concentrations (MICs) of 6 biocides (sodium hypochlorite, chlorhexidine digluconate, benzalkonium chloride, povidone-iodine, ethanol and triclosan) were determined in triplicate at 25°C and 37°C in Mueller-Hinton broth (MHB) and M9 minimum medium, against 17 CP-Kp isolates representing different clones (HR and no-HR), sequence-types (STs) and carbapenemases. Efflux pumps genes were detected by whole genome sequencing (MiSeq). RESULTS: Median MICs were slightly higher at 37°C than at 25°C (p≤0.05), except for benzalkonium chloride, triclosan and ethanol. MIC medians were much higher in MHB than in M9, except for triclosan. No significant differences were observed in the median MICs, regarding the type of clone, ST or carbapenemase; cepA, acrAB, kpnEF and oqxAB genes were detected in all isolates, whereas qacE and qacA were not detected; smvAR, and qacΔE genes were detected in 94% and 47% of isolates, respectively. CONCLUSIONS: Triclosan, chlorhexidine digluconate, benzalkonium chloride and ethanol were the most active biocides. The activity of some biocides is affected by temperature and growth media, suggesting that standardised procedures for biocide susceptibility testing based on MIC determination are required. This activity, in terms of MICs, are not related to the type of clone, ST, carbapenemase or the presence of the efflux pump genes.

Misidentification of the SARS-CoV-2 Mu variant using commercial mutation screening assays.

Detection of mutations by multiplex real-time RT-PCR is a widely used method for the screening of SARS-CoV-2 variants, but this method has several limitations. We describe three cases in which a Mu strain containing the mutation K417N was initially misclassified as the Beta variant. We recommend the detection of P681H to distinguish between these two variants. Our experience highlights the importance of keeping track of new variants and mutations in order to adapt the current workflows.

Transfer of plasmids harbouring blaOXA-48-like carbapenemase genes in biofilm-growing Klebsiella pneumoniae: Effect of biocide exposure.

The spread of OXA-48-encoding plasmids from Klebsiella pneumoniae (OXA-48-Kpn), especially successful high-risk (HR) clones, is a growing concern. Biofilm formation can contribute to the dissemination of OXA-48-Kpn. It is not known whether biocides can affect the transfer of OXA-48-Kpn in biofilm. The aim of this study was to evaluate the effect of biocides on the conjugation frequency (CF) of OXA-48-Kpn in both biofilm and planktonic cultures. For that, seven OXA-48-Kpn isolates (4 belonging to HR clones and 3 to non-HR clones) were selected as donors. Each isolate was mixed (1:1) with Escherichia coli J53 (recipient) and grown on polystyrene microplates without biocides (control) and with 0.25x MIC of triclosan (TRI), chlorhexidine digluconate (CHX), povidone-iodine (POV), sodium hypochlorite (SOD) or ethanol (ETH). The CF was calculated as the number of transconjugants/number of E. coli J53. The results showed that for isolates growing in the absence of biocide, the mean fold change in the CF in biofilm with respect to that determined in planktonic cells (CF-BF/CF-PK) was 0.2 in non-HR isolates and ranged from 2.0 to 14.7 in HR isolates. In HR isolates grown in the presence of biocide, especially CHX, TRI, and ETH, the fold changes in CF-BF/CF-PK decreased, whereas in non-HR isolates the fold changes were similar or increased slightly with CHX, ETH, SOD and POV. In conclusion, the fold changes in the CF-BF/CF-PK are higher in HR isolates comparing to non-HR isolates in abscence of biocides. The fold changes in CF-BF/CF-PK of the HR isolates in the presence of biocides varied with the type of biocides, whereas in non-HR isolates, biocides have no significant effect, or produce only a slight increase in the fold change of CF-BF/CF-PK.

Co-transfer of plasmid-encoded bla carbapenemases genes and mercury resistance operon in high-risk clones of Klebsiella pneumoniae.

Carbapenemase-producing Klebsiella pneumoniae (CP-Kp) is a real global health threat. Environmental reservoirs of resistance gene determinats, such as effluents of hospital wastewaters, are acquiring increased relevance in the selection of plasmid-encoded carbapenemase genes. The presence of Hg in environmental reservoirs may exert a positive selective pressure on tolerant bacteria, favoring the co-transfer of carbapenemase genes and mer operons. In our study, 63 CP-Kp isolates were screened for mer operons by whole genome sequencing (MySeq). Conjugation assays were performed with 24 out of 63 CP-Kp isolates harboring the mer operon. Ten transconjugants (Tc-Kp) were selected with Hg. Plasmid DNA of Tc-Kp was extracted and sequenced using single-molecule real-time (SMRT) technology (PacBio, Sequel II system) with later annotation. Plasmid analysis revealed that Tc-Kp from blaIMP-like (n = 3) showed a single plasmid belonging to IncC group with two complete mer operon next to blaIMP-like. Tc-Kp from blaVIM-1 (n = 2) harbored two plasmids, one with blaVIM-1 in an IncL, and mer operon was in an IncFIB plasmid. Tc-Kp from blaOXA-48-like (n = 5) showed 2 plasmids. blaOXA-48-like was found in an IncL plasmid, whereas mer operon was (i) in an IncR plasmid associated with blaCTX-M-15 in 3 Tc-Kp-OXA-48-like, (ii) in an IncC plasmid associated with blaCMY-2 in 1 Tc-Kp-OXA-48-like, (iii) and in an IncFIB plasmid associated with blaCTX-M-15 in 1 Tc-Kp-OXA-48-like. This is, to our knowledge, the first study to describe in K. pneumoniae producing plasmid-encoded carbapenemase, the potential impact of Hg in the co-transfer of mer operons and carbapenemase genes located in the same or different plasmids. KEY POINTS: • Environmental reservoirs are playing an important role in the selection of carbapenemase genes. • Conjugation assays, selecting with Hg, obtained 10 transconjugants with carbapenemase genes. • mer operons were located in the same or different plasmids than carbapenemase genes.

In vitro activity of six biocides against carbapenemase-producing Klebsiella pneumoniae and presence of genes encoding efflux pumps.

INTRODUCTION: Acquisition of reduced susceptibility to biocides may contribute to the dissemination of high-risk (HR) clones of carbapenemase-producing Klebsiella pneumoniae (CP-Kp). The aim of this study was (a) to determinate the activity of biocides against CP-Kp, and (b) to analyse the relationship between biocide activity and the presence of efflux pumps. METHODS: The minimal inhibitory concentrations (MICs) of 6 biocides (sodium hypochlorite, chlorhexidine digluconate, benzalkonium chloride, povidone-iodine, ethanol and triclosan) were determined in triplicate at 25°C and 37°C in Mueller-Hinton broth (MHB) and M9 minimum medium, against 17 CP-Kp isolates representing different clones (HR and no-HR), sequence-types (STs) and carbapenemases. Efflux pumps genes were detected by whole genome sequencing (MiSeq). RESULTS: Median MICs were slightly higher at 37°C than at 25°C (p≤0.05), except for benzalkonium chloride, triclosan and ethanol. MIC medians were much higher in MHB than in M9, except for triclosan. No significant differences were observed in the median MICs, regarding the type of clone, ST or carbapenemase; cepA, acrAB, kpnEF and oqxAB genes were detected in all isolates, whereas qacE and qacA were not detected; smvAR, and qacΔE genes were detected in 94%and 47% of isolates, respectively. CONCLUSIONS: Triclosan, chlorhexidine digluconate, benzalkonium chloride and ethanol were the most active biocides. The activity of some biocides is affected by temperature and growth media, suggesting that standardised procedures for biocide susceptibility testing based on MIC determination are required. This activity, in terms of MICs, are not related to the type of clone, ST, carbapenemase or the presence of the efflux pump genes.

Multicenter evaluation of the Panbio™ COVID-19 rapid antigen-detection test for the diagnosis of SARS-CoV-2 infection.

OBJECTIVES: The standard RT-PCR assay for coronavirus disease 2019 (COVID-19) is laborious and time-consuming, limiting testing availability. Rapid antigen-detection tests are faster and less expensive; however, the reliability of these tests must be validated before they can be used widely. The objective of this study was to determine the performance of the Panbio™ COVID-19 Ag Rapid Test Device (PanbioRT) (Abbott) in detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in nasopharyngeal swab specimens. METHODS: This prospective multicentre study was carried out in ten Spanish university hospitals and included individuals with clinical symptoms or epidemiological criteria of COVID-19. Only individuals with ≤7 days from the onset of symptoms or from exposure to a confirmed case of COVID-19 were included. Two nasopharyngeal samples were taken to perform the PanbioRT as a point-of-care test and a diagnostic RT-PCR test. RESULTS: Among the 958 patients studied, 325 (90.5%) had true-positive results. The overall sensitivity and specificity for the PanbioRT were 90.5% (95%CI 87.5-93.6) and 98.8% (95%CI 98-99.7), respectively. Sensitivity in participants who had a threshold cycle (CT) < 25 for the RT-PCR test was 99.5% (95%CI 98.4-100), and in participants with ≤5 days of the clinical course it was 91.8% (95%CI 88.8-94.8). Agreement between techniques was 95.7% (κ score 0.90; 95%CI 0.88-0.93). CONCLUSIONS: The PanbioRT performs well clinically, with even more reliable results for patients with a shorter clinical course of the disease or a higher viral load. The results must be interpreted based on the local epidemiological context.

Infección de prótesis articular por Peptostreptococcus canis y Bacteroides pyogenes / Peptostreptococcus canis and Bacteroides pyogenes prosthetic joint infection

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