Since their discovery, toxin-antitoxin (TA) systems have captivated the attention of many scientists. Recent studies have demonstrated that TA systems play a key role in phage inhibition. The aim of ...the present study was to investigate the role of the PemIK (PemK/PemI) type II TA system in phage inhibition by its intrinsic expression in clinical strains of Klebsiella pneumoniae carrying the lncL plasmid, which harbours the carbapenemase OXA-48 and the PemK/PemI TA system. Furthermore, induced expression of the system in an IPTG-inducible plasmid in a reference strain of K. pneumoniae ATCC10031 was also studied. The results showed that induced expression of the whole TA system did not inhibit phage infection, whereas overexpression of the pemK toxin prevented early infection. To investigate the molecular mechanism involved in the PemK toxin-mediated inhibition of phage infection, assays measuring metabolic activity and viability were performed, revealing that overexpression of the PemK toxin led to dormancy of the bacteria. Thus, we demonstrate that the PemK/PemI TA system plays a role in phage infection and that the action of the free toxin induces a dormant state in the cells, resulting in inhibition of phage infections.
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
bla
IMP-like
(
n
= 3) showed a single plasmid belonging to IncC group with two complete
mer
operon next to
bla
IMP-like
. Tc-Kp from
bla
VIM-1
(n = 2) harbored two plasmids, one with
bla
VIM-1
in an IncL, and
mer
operon was in an IncFIB plasmid. Tc-Kp from
bla
OXA-48-like
(
n
= 5) showed 2 plasmids.
bla
OXA-48-like
was found in an IncL plasmid, whereas
mer
operon was (i) in an IncR plasmid associated with
bla
CTX-M-15
in 3 Tc-Kp-OXA-48-like, (ii) in an IncC plasmid associated with
bla
CMY-2
in 1 Tc-Kp-OXA-48-like, (iii) and in an IncFIB plasmid associated with
bla
CTX-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.
Graphical abstract
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.
Phages are increasingly considered promising alternatives to target drug-resistant bacterial pathogens. However, their often-narrow host range can make it challenging to find matching phages against ...bacteria of interest. Current computational tools do not accurately predict interactions at the strain level in a way that is relevant and properly evaluated for practical use. We present PhageHostLearn, a machine learning system that predicts strain-level interactions between receptor-binding proteins and bacterial receptors for Klebsiella phage-bacteria pairs. We evaluate this system both in silico and in the laboratory, in the clinically relevant setting of finding matching phages against bacterial strains. PhageHostLearn reaches a cross-validated ROC AUC of up to 81.8% in silico and maintains this performance in laboratory validation. Our approach provides a framework for developing and evaluating phage-host prediction methods that are useful in practice, which we believe to be a meaningful contribution to the machine-learning-guided development of phage therapeutics and diagnostics.
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.
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.
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).
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.
Infections caused by carbapenem-resistant
are a global threat causing a high number of fatal infections. This microorganism can also easily acquire antibiotic resistance determinants, making the ...treatment of infections a big challenge, and has the ability to persist in the hospital environment under a wide range of conditions. The objective of this work was to study the molecular epidemiology and genetic characteristics of two
outbreaks (2009 and 2020-21) at a tertiary hospital in Northern Spain. Thirty-six isolates were investigated and genotypically screened by Whole Genome Sequencing to analyse the resistome and virulome. Isolates were resistant to carbapenems, aminoglycosides and fluoroquinolones. Multi-Locus Sequence Typing analysis identified that Outbreak 1 was mainly produced by isolates belonging to ST3
/ST106
(IC3) containing
,
and
. Outbreak 2 isolates were exclusively ST2
/ST801
(IC2)
,
and
, the same genotype seen in two isolates from 2009. Virulome analysis showed that IC2 isolates contained genes for capsular polysaccharide KL32 and lipooligosacharide OCL5. A 8.9 Kb plasmid encoding the
gene was common in all isolates. The persistance over time of a virulent IC2 clone highlights the need of active surveillance to control its spread.
To describe participant characteristics associated with severe acute respiratory syndrome coronavirus 2 infection in Spain's first 2 COVID-19 waves per the Spanish National Seroepidemiological Survey ...of SARS-CoV-2 Infection (ENE-COVID).
A representative cohort of the noninstitutionalized Spanish population, selected through stratified 2-stage sampling, answered a questionnaire and received point-of-care testing April to June 2020 (first wave: n = 68 287); previously seronegative participants repeated the questionnaire and test November 2020 (second wave: n = 44 451). We estimated seropositivity by wave and participant characteristics, accounting for sampling weights, nonresponse, and design effects.
We found that 6.0% (95% confidence interval CI = 5.7%, 6.4%) of Spain's population was infected by June and 3.8% (95% CI = 3.5%, 4.1%) more by November 2020. Both genders were equally affected. Seroprevalence decreased with age in adults 20 years and older in the second wave; socioeconomic differences increased. Health care workers were affected at 11.1% (95% CI = 9.0%, 13.6%) and 6.1% (95% CI = 4.4%, 8.5%) in the first and second waves, respectively. Living with an infected person increased infection risk to 22.1% (95% CI = 18.9%, 25.6%) in the first and 35.0% (95% CI = 30.8%, 39.4%) in the second wave.
ENE-COVID characterized the first 2 pandemic waves, when information from surveillance systems was incomplete. (
. 2023;113(5):533-544. https://doi.org/10.2105/AJPH.2023.307233).
Fourier-transform infrared (FTIR) spectroscopy has the potential to be used for bacterial typing and outbreak characterization. We evaluated FTIR for the characterization of an outbreak caused by
...Elizabethkingia miricola
. During the 2020–2021 period, 26 isolates (23 clinical and 3 environmental) were collected and analyzed by FTIR (IR Biotyper) and core-genome MLST (cgMLST), in addition to antimicrobial susceptibility testing. FTIR spectroscopy and cgMLST showed that 22 of the isolates were related to the outbreak, including the environmental samples, with only one discordance between both methods. Then, FTIR is useful for
E. miricola
typing and can be easily implemented in the laboratory.
•Emerge of linezolid-resistant Enterococcus faecalis carrying optrA in Spain.•High dissemination capability of optrA-carrying E. faecalis belonging to ST585 and ST480.•Co-existence of optrA and poxtA ...plasmid-borne genes in a clinical setting.
The emergence of linezolid-resistant Enterococcus spp. (LRE) due to transferable resistance determinants is a matter of concern. To understand the contribution of the plasmid-encoded optrA and poxtA genes to the emergence of LRE, clinical isolates from different Spanish hospitals submitted to the Spanish Reference Laboratory from 2015–2018 were analysed. Linezolid resistance mechanisms were screened in all isolates by PCR and sequencing. Genetic relatedness of Enterococcus spp. carrying optrA and poxtA was studied by PFGE and MLST. Antimicrobial susceptibility was tested by broth microdilution using EUCAST standards. A total of 97 LRE isolates were studied, in 94 (96.9%) of which at least one resistance determinant was detected; 84/97 isolates (86.6%) presented a single resistance mechanism as follows: 45/84 (53.6%) carried the optrA gene, 38/84 (45.2%) carried the G2576T mutation and 1/84 (1.2%) carried the poxtA gene. In addition, 5/97 isolates (5.2%) carried both optrA and the G2576T mutation and 5/97 (5.2%) carried both optrA and poxtA. The optrA gene was more frequent in Enterococcus faecalis (83.6%) than Enterococcus faecium (11.1%) and was mainly associated with community-acquired urinary tract infections. Carriage of the poxtA gene was more frequent in E. faecium (13.9%) than E. faecalis (1.6%). Among the optrA-positive E. faecalis isolates, two main clusters were detected by PFGE. These two clusters belonged to ST585 and ST480 and were distributed throughout 11 and 6 Spanish provinces, respectively. This is the first description of LRE carrying the poxtA gene in Spain, including the co-existence of optrA and poxtA in five isolates.
Mucins are important glycoproteins that form a protective layer throughout the gastrointestinal and respiratory tracts. There is scientific evidence of increase in phage-resistance in the presence of ...mucin for some bacterial pathogens. Manipulation in mucin composition may ultimately influence the effectiveness of phage therapy. In this work, two clinical strains of
K. pneumoniae
(K3574 and K3325), were exposed to the lytic bacteriophage vB_KpnS-VAC35 in the presence and absence of mucin on a long-term co-evolution assay, in an attempt to mimic
in vitro
the exposure to mucins that bacteria and their phages face
in vivo
. Enumerations of the bacterial and phage counts at regular time intervals were conducted, and extraction of the genomic DNA of co-evolved bacteria to the phage, the mucin and both was performed. We determined the frequency of phage-resistant mutants in the presence and absence of mucin and including a mucolytic agent (N-acetyl L-cysteine, NAC), and sequenced them using Nanopore. We phenotypically demonstrated that the presence of mucin induces the emergence of bacterial resistance against lytic phages, effectively decreased in the presence of NAC. In addition, the genomic analysis revealed some of the genes relevant to the development of phage resistance in long-term co-evolution, with a special focus on the mucoid environment. Genes involved in the metabolism of carbohydrates were mutated in the presence of mucin. In conclusion, the use of mucolytic agents prior to the administration of lytic phages could be an interesting therapeutic option when addressing
K. pneumoniae
infections in environments where mucin is overproduced.
Extended-spectrum ß-lactamase-producing Enterobacterales (ESBL-E) are a serious threat among emerging antibiotic resistant bacteria. Particularly, the number of cases of ESBL-E infections reported in ...children has been increasing in recent years, and approved antibiotic treatments for this age group are limited. However, information regarding the prevalence of colonization in European children, risk factors associated with colonization, and the characteristics of the colonizing strains is scarce. The aims of this study were to determine the prevalence of ESBL-E colonization in fecal samples of apparently healthy schoolchildren, to identify lifestyle routines associated with colonization, and to characterize clonal relationships and mechanisms of resistance in ESBL-E isolates.
A cohort of 887 healthy children (3-13 years old) from seven primary and secondary schools in the Madrid metropolitan area was recruited between April-June 2018, and sociodemographic information and daily habits were collected. Fecal samples were screened for ESBL-E carriage in selective medium. ESBL-E isolates were further characterized by assessing molecular epidemiology (PFGE and MLST), ESBL gene carriage, and antibiotic resistance profile. This information was analyzed in conjunction with the metadata of the participants in order to identify external factors associated with ESBL-E carriage.
Twenty four ESBL-E, all but one
were detected in 23 children (prevalence: 2.6%; 95% CI: 1.6-3.6%). Of these, seven contained the
allele, five the
, five the
, three the
, three the
, and one the
. Significant clonal diversity was observed among the isolates that grouped into 22 distinct clusters (at <85% similarity of PFGE profile). ESBL-producing
isolates belonged to 12 different STs, with ST10 (25%) and ST131 (17%) being the most frequent. Apart from ß-lactams, resistance to trimethoprim/sulfamethoxazole (46%), ciprofloxacin (33%), levofloxacin (33%), tobramycin (21%), and gentamicin (8%) were the most frequently detected.
The prevalence of ESBL-E in the studied cohort of children was lower than the average colonization rate previously detected in Europe for both children and adults.
was the main ESBL-producing species detected and CTX-M were the most frequently identified ESBLs. High ST diversity suggests polyclonal dissemination. Compared to other STs, ST131 isolates were associated with resistance to various antimicrobials.
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