Genomic context as well as sequence of both psr and penicillin-binding protein 5 contributes to ß-lactam resistance in Enterococcus faecium.

Penicillin-binding protein 5 (PBP5) of Enterococcus faecium (Efm) is vital for ampicillin resistance (AMP-R). We previously designated three forms of PBP5, namely, PBP5-S in Efm clade B strains [ampicillin susceptible (AMP-S)], PBP5-S/R (AMP-S or R), and PBP5-R (AMP-R) in clade A strains. Here, pbp5 deletion resulted in a marked reduction in AMP minimum inhibitory concentrations (MICs) to 0.01-0.09 µg/mL for clade B and 0.12-0.19 µg/mL for clade A strains; in situ complementation restored parental AMP MICs. Using D344SRF (lacking ftsW/psr/pbp5), constructs with ftsWA/psrA (from a clade A1 strain) cloned upstream of pbp5-S and pbp5-S/R alleles resulted in modest increases in MICs to 3-8 µg/mL, while high MICs (>64 µg/mL) were seen using pbp5 from A1 strains. Next, using ftsW ± psr from clade B and clade A/B and B/A hybrid constructs, the presence of psrB, even alone or in trans, resulted in much lower AMP MICs (3-8 µg/mL) than when psrA was present (MICs >64 µg/mL). qRT PCR showed relatively greater pbp5 expression (P = 0.007) with pbp5 cloned downstream of clade A1 ftsW/psr (MIC >128 µg/mL) vs when cloned downstream of clade B ftsW/psr (MIC 4-16 µg/mL), consistent with results in western blots. In conclusion, we report the effect of clade A vs B psr on AMP MICs as well as the impact of pbp5 alleles from different clades. While previously, Psr was not thought to contribute to AMP MICs in Efm, our results showed that the presence of psrB resulted in a major decrease in Efm AMP MICs. IMPORTANCE: The findings of this study shed light on ampicillin resistance in Enterococcus faecium clade A strains. They underscore the significance of alterations in the amino acid sequence of penicillin-binding protein 5 (PBP5) and the pivotal role of the psr region in PBP5 expression and ampicillin resistance. Notably, the presence of a full-length psrB leads to reduced PBP5 expression and lower minimum inhibitory concentrations (MICs) of ampicillin compared to the presence of a shorter psrA, regardless of the pbp5 allele involved. Additionally, clade B E. faecium strains exhibit lower AMP MICs when both psr alleles from clades A and B are present, although it is important to consider other distinctions between clade A and B strains that may contribute to this effect. It is intriguing to note that the divergence between clade A and clade B E. faecium and the subsequent evolution of heightened AMP MICs in hospital-associated strains appear to coincide with changes in Pbp5 and psr. These changes in psr may have resulted in an inactive Psr, facilitating increased PBP5 expression and greater ampicillin resistance. These results raise the possibility that a mimicker of PsrB, if one could be designed, might be able to lower MICs of ampicillin-resistant E. faecium, thus potentially resorting ampicillin to our therapeutic armamentarium for this species.

Enterococcus faecium Clade Competition in the Presence of ß-Lactam Antibiotics in a Mouse GI Tract Colonization Model.

Previously, we showed that Enterococcus faecium clade B strains outcompeted health care-associated clade A1 strains in murine gastrointestinal colonization. Here, parenterally administered piperacillin-tazobactam and ceftriaxone significantly promoted colonization by clade A1 over clade B strains except that ceftriaxone, at the dose used, did not favor the least ß-lactam-resistant A1 strain. The advantage that ß-lactam administration gives to more highly ampicillin-resistant E. faecium over ampicillin-susceptible strains mirrors what occurs in hospitalized patients administered these antibiotics.

Drinking water chlorination has minor effects on the intestinal flora and resistomes of Bangladeshi children.

Healthy development of the gut microbiome provides long-term health benefits. Children raised in countries with high infectious disease burdens are frequently exposed to diarrhoeal pathogens and antibiotics, which perturb gut microbiome assembly. A recent cluster-randomized trial leveraging >4,000 child observations in Dhaka, Bangladesh, found that automated water chlorination of shared taps effectively reduced child diarrhoea and antibiotic use. In this substudy, we leveraged stool samples collected from 130 children 1 year after chlorine doser installation to examine differences between treatment and control children's gut microbiota. Water chlorination was associated with increased abundance of several bacterial genera previously linked to improved gut health; however, we observed no effects on the overall richness or diversity of taxa. Several clinically relevant antibiotic resistance genes were relatively more abundant in the gut microbiome of treatment children, possibly due to increases in Enterobacteriaceae. While further studies on the long-term health impacts of drinking chlorinated water would be valuable, we conclude that access to chlorinated water did not substantially impact child gut microbiome development in this setting, supporting the use of chlorination to increase global access to safe drinking water.

Digital multiplex ligation assay for highly multiplexed screening of ß-lactamase-encoding genes in bacterial isolates.

Increasing incidence of antibiotic resistance in clinical and environmental settings calls for increased scalability in their surveillance. Current screening technologies are limited by the number of samples and genes that can easily be screened. We demonstrate here digital multiplex ligation assay (dMLA) as a low-cost targeted genomic detection workflow capable of highly-parallel screening of bacterial isolates for multiple target gene regions simultaneously. Here, dMLA is used for simultaneous detection of 1187 ß-lactamase-encoding genes, including extended spectrum ß-lactamase (ESBL) genes, in 74 bacterial isolates. We demonstrate dMLA as a light-weight and cost-efficient workflow which provides a highly scalable tool for antimicrobial resistance surveillance and is also adaptable to genetic screening applications beyond antibiotic resistance.

Urban informal settlements as hotspots of antimicrobial resistance and the need to curb environmental transmission.

Antimicrobial resistance (AMR) is a growing public health challenge that is expected to disproportionately burden lower- and middle-income countries (LMICs) in the coming decades. Although the contributions of human and veterinary antibiotic misuse to this crisis are well-recognized, environmental transmission (via water, soil or food contaminated with human and animal faeces) has been given less attention as a global driver of AMR, especially in urban informal settlements in LMICs-commonly known as 'shanty towns' or 'slums'. These settlements may be unique hotspots for environmental AMR transmission given: (1) the high density of humans, livestock and vermin living in close proximity; (2) frequent antibiotic misuse; and (3) insufficient drinking water, drainage and sanitation infrastructure. Here, we highlight the need for strategies to disrupt environmental AMR transmission in urban informal settlements. We propose that water and waste infrastructure improvements tailored to these settings should be evaluated for their effectiveness in limiting environmental AMR dissemination, lowering the community-level burden of antimicrobial-resistant infections and preventing antibiotic misuse. We also suggest that additional research is directed towards developing economic and legal incentives for evaluating and implementing water and waste infrastructure in these settings. Given that almost 90% of urban population growth will occur in regions predicted to be most burdened by the AMR crisis, there is an urgent need to build effective, evidence-based policies that could influence massive investments in the built urban environment in LMICs over the next few decades.

High Genomic Diversity and Heterogenous Origins of Pathogenic and Antibiotic-Resistant Escherichia coli in Household Settings Represent a Challenge to Reducing Transmission in Low-Income Settings.

Escherichia coli is present in multiple hosts and environmental compartments as a normal inhabitant, temporary or persistent colonizer, and as a pathogen. Transmission of E. coli between hosts and with the environment is considered to occur more often in areas with poor sanitation. We performed whole-genome comparative analyses on 60 E. coli isolates from soils and fecal sources (cattle, chickens, and humans) in households in rural Bangladesh. Isolates from household soils were in multiple branches of the reconstructed phylogeny, intermixed with isolates from fecal sources. Pairwise differences between all strain pairs were large (minimum, 189 single nucleotide polymorphisms [SNPs]), suggesting high diversity and heterogeneous origins of the isolates. The presence of multiple virulence and antibiotic resistance genes is indicative of the risk that E. coli from soil and feces represent for the transmission of variants that pose potential harm to people. Analysis of the accessory genomes of the Bangladeshi E. coli relative to E. coli genomes available in NCBI identified a common pool of accessory genes shared among E. coli isolates in this geographic area. Together, these findings indicate that in rural Bangladesh, a high level of E. coli in soil is likely driven by contributions from multiple and diverse E. coli sources (human and animal) that share an accessory gene pool relatively unique to previously published E. coli genomes. Thus, interventions to reduce environmental pathogen or antimicrobial resistance transmission should adopt integrated One Health approaches that consider heterogeneous origins and high diversity to improve effectiveness and reduce prevalence and transmission.IMPORTANCEEscherichia coli is reported in high levels in household soil in low-income settings. When E. coli reaches a soil environment, different mechanisms, including survival, clonal expansion, and genetic exchange, have the potential to either maintain or generate E. coli variants with capabilities of causing harm to people. In this study, we used whole-genome sequencing to identify that E. coli isolates collected from rural Bangladeshi household soils, including pathogenic and antibiotic-resistant variants, are diverse and likely originated from multiple diverse sources. In addition, we observed specialization of the accessory genome of this Bangladeshi E. coli compared to E. coli genomes available in current sequence databases. Thus, to address the high level of pathogenic and antibiotic-resistant E. coli transmission in low-income settings, interventions should focus on addressing the heterogeneous origins and high diversity.

Risk Factors for Detection, Survival, and Growth of Antibiotic-Resistant and Pathogenic Escherichia coli in Household Soils in Rural Bangladesh.

Soils in household environments in low- and middle-income countries may play an important role in the persistence, proliferation, and transmission of Escherichia coli Our goal was to investigate the risk factors for detection, survival, and growth of E. coli in soils collected from household plots. E. coli was enumerated in soil and fecal samples from humans, chickens, and cattle from 52 households in rural Bangladesh. Associations between E. coli concentrations in soil, household-level risk factors, and soil physicochemical characteristics were investigated. Susceptibility to 16 antibiotics and the presence of intestinal pathotypes were evaluated for 175 E. coli isolates. The growth and survival of E. coli in microcosms using soil collected from the households were also assessed. E. coli was isolated from 44.2% of the soil samples, with an average of 1.95 log10 CFU/g dry soil. Soil moisture and clay content were associated with E. coli concentrations in soil, whereas no household-level risk factor was significantly correlated. Antibiotic resistance and pathogenicity were common among E. coli isolates, with 42.3% resistant to at least one antibiotic, 12.6% multidrug resistant (≥3 classes), and 10% potentially pathogenic. Soil microcosms demonstrate growth and/or survival of E. coli, including an enteropathogenic extended-spectrum beta-lactamase (ESBL)-producing isolate, in some, but not all, of the household soils tested. In rural Bangladesh, defined soil physicochemical characteristics appear more influential for E. coli detection in soils than household-level risk factors. Soils may act as reservoirs in the transmission of antibiotic-resistant and potentially pathogenic E. coli and therefore may impact the effectiveness of water, sanitation, and hygiene interventions.IMPORTANCE Soil may represent a direct source or act as an intermediary for the transmission of antibiotic-resistant and pathogenic Escherichia coli strains, particularly in low-income and rural settings. Thus, determining risk factors associated with detection, growth, and long-term survival of E. coli in soil environments is important for public health. Here, we demonstrate that household soils in rural Bangladesh are reservoirs for antibiotic-resistant and potentially pathogenic E. coli strains and can support E. coli growth and survival, and defined soil physicochemical characteristics are drivers of E. coli survival in this environment. In contrast, we found no evidence that household-level factors, including water, sanitation, and hygiene indicators, were associated with E. coli contamination of household soils.

Escherichia coli Contamination across Multiple Environmental Compartments (Soil, Hands, Drinking Water, and Handwashing Water) in Urban Harare: Correlations and Risk Factors.

Escherichia coli pathotypes (i.e., enteropathogenic and enterotoxigenic) have been identified among the pathogens most responsible for moderate-to-severe diarrhea in low- and middle-income countries (LMICs). Pathogenic E. coli are transmitted from infected human or animal feces to new susceptible hosts via environmental reservoirs such as hands, water, and soil. Commensal E. coli, which includes nonpathogenic E. coli strains, are widely used as fecal bacteria indicator, with their presence associated with increased likelihood of enteric pathogens and/or diarrheal disease. In this study, we investigated E. coli contamination in environmental reservoirs within households (N = 142) in high-population density communities of Harare, Zimbabwe. We further assessed the interconnectedness of the environmental compartments by investigating associations between, and household-level risk factors for, E. coli contamination. From the data we collected, the source and risk factors for E. coli contamination are not readily apparent. One notable exception is the presence of running tap water on the household plot, which is associated with significantly less E. coli contamination of drinking water, handwashing water, and hands after handwashing. In addition, E. coli levels on hands after washing are significantly associated with handwashing water contamination, hand contamination before washing, and diarrhea incidence. Finally, we observed that animal ownership increases E. coli contamination in soil, and E. coli in soil are correlated with contamination on hands before washing. This study highlights the complexity of E. coli contamination in household environments within LMICs. More, larger, studies are needed to better identify sources and exposure pathways of E. coli-and enteric pathogens generally-to identify effective interventions.

Processing of the major autolysin of E. faecalis, AtlA, by the zinc-metalloprotease, GelE, impacts AtlA septal localization and cell separation.

AtlA is the major peptidoglycan hydrolase of Enterococcus faecalis involved in cell division and cellular autolysis. The secreted zinc metalloprotease, gelatinase (GelE), has been identified as an important regulator of cellular function through post-translational modification of protein substrates. AtlA is a known target of GelE, and their interplay has been proposed to regulate AtlA function. To study the protease-mediated post-translational modification of AtlA, monoclonal antibodies were developed as research tools. Flow cytometry and Western blot analysis suggests that in the presence of GelE, surface-bound AtlA exists primarily as a N-terminally truncated form whereas in the absence of GelE, the N-terminal domain of AtlA is retained. We identified the primary GelE cleavage site occurring near the transition between the T/E rich Domain I and catalytic region, Domain II via N-terminal sequencing. Truncation of AtlA had no effect on the peptidoglycan hydrolysis activity of AtlA. However, we observed that N-terminal cleavage was required for efficient AtlA-mediated cell division while unprocessed AtlA was unable to resolve dividing cells into individual units. Furthermore, we observed that the processed AtlA has the propensity to localize to the cell septum on wild-type cells whereas unprocessed AtlA in the ΔgelE strain were dispersed over the cell surface. Combined, these results suggest that AtlA septum localization and subsequent cell separation can be modulated by a single GelE-mediated N-terminal cleavage event, providing new insights into the post-translation modification of AtlA and the mechanisms governing chaining and cell separation.

Differential Penicillin-Binding Protein 5 (PBP5) Levels in the Enterococcus faecium Clades with Different Levels of Ampicillin Resistance.

Ampicillin resistance in Enterococcus faecium is a serious concern worldwide, complicating the treatment of E. faecium infections. Penicillin-binding protein 5 (PBP5) is considered the main ampicillin resistance determinant in E. faecium The three known E. faecium clades showed sequence variations in the pbp5 gene that are associated with their ampicillin resistance phenotype; however, these changes alone do not explain the array of resistance levels observed among E. faecium clinical strains. We aimed to determine if the levels of PBP5 are differentially regulated between the E. faecium clades, with the hypothesis that variations in PBP5 levels could help account for the spectrum of ampicillin MICs seen in E. faecium We studied pbp5 mRNA levels and PBP5 protein levels as well as the genetic environment upstream of pbp5 in 16 E. faecium strains that belong to the different E. faecium clades and for which the ampicillin MICs covered a wide range. Our results found that pbp5 and PBP5 levels are increased in subclade A1 and A2 ampicillin-resistant strains compared to those in clade B and subclade A2 ampicillin-susceptible strains. Furthermore, we found evidence of major clade-associated rearrangements in the region upstream of pbp5, including large DNA fragment insertions, deletions, and single nucleotide polymorphisms, that may be associated with the differential regulation of PBP5 levels between the E. faecium clades. Overall, these findings highlight the contribution of the clade background to the regulation of PBP5 abundance and point to differences in the region upstream of pbp5 as likely contributors to the differential expression of ampicillin resistance.

Enterococcus faecalis Ebp pili are important for cell-cell aggregation and intraspecies gene transfer.

Enterococcus faecalis is an opportunistic pathogen that ranks among the leading causes of biofilm-associated infections. We previously demonstrated that the endocarditis- and biofilm-associated pili (Ebp) of E. faecalis play a major role in biofilm formation, adherence to abiotic surfaces and experimental infections. In this study, derivatives of E. faecalis strain OG1 were engineered to further characterize functions of Ebp pili. Loss of pili resulted in a 36-fold decrease in the number of closely associated cells when OG1RFΔebpABC was mixed with OG1SSpΔebpABC, compared with mixing the Ebp+ parental strains. In addition, using the Ebp+ parental strains as donor and recipient, we found a statistically significant increase (280-360 %, P < 0.05) in the frequency of plasmid transfer versus using Ebp- mutants in the conjugation experiments. These results demonstrate a previously unrecognized role of Ebp pili, namely, as important contributors to microscale cell aggregation and horizontal spread of genetic material.

Role of the Emp Pilus Subunits of Enterococcus faecium in Biofilm Formation, Adherence to Host Extracellular Matrix Components, and Experimental Infection.

Enterococcus faecium is an important cause of hospital-associated infections, including urinary tract infections (UTIs), bacteremia, and infective endocarditis. Pili have been shown to play a role in the pathogenesis of Gram-positive bacteria, including E. faecium We previously demonstrated that a nonpiliated ΔempABC::cat derivative of E. faecium TX82 was attenuated in biofilm formation and in a UTI model. Here, we studied the contributions of the individual pilus subunits EmpA, EmpB, and EmpC to pilus architecture, biofilm formation, adherence to extracellular matrix (ECM) proteins, and infection. We identified EmpA as the tip of the pili and found that deletion of empA reduced biofilm formation to the same level as deletion of the empABC operon, a phenotype that was restored by reconstituting in situ the empA gene. Deletion of empB also caused a reduction in biofilm, while EmpC was found to be dispensable. Significant reductions in adherence to fibrinogen and collagen type I were observed with deletion of empA and empB, while deletion of empC had no adherence defect. Furthermore, we showed that each deletion mutant was significantly attenuated in comparison to the isogenic parental strain, TX82, in a mixed-inoculum UTI model (P < 0.001 to 0.048), that reconstitution of empA restored virulence in the UTI model, and that deletion of empA also resulted in attenuation in an infective endocarditis model (P = 0.0088). Our results indicate that EmpA and EmpB, but not EmpC, contribute to biofilm and adherence to ECM proteins; however, all the Emp pilins are important for E. faecium to cause infection in the urinary tract.

Gastrointestinal Tract Colonization Dynamics by Different Enterococcus faecium Clades.

Colonization of the gastrointestinal tract (GIT) generally precedes infection with antibiotic-resistant Enterococcus faecium We used a mouse GIT colonization model to test differences in the colonization levels by strains from different E. faecium lineages: clade B, part of the healthy human microbiota; subclade A1, associated with infections; and subclade A2, primarily associated with animals. After mono-inoculation, there was no significant difference in colonization (measured as the geometric mean number of colony-forming units per gram) by the E. faecium clades at any time point (P > .05). However, in competition assays, with 6 of the 7 pairs, clade B strains outcompeted clade A strains in their ability to persist in the GIT; this difference was significant in some pairs by day 2 and in all pairs by day 14 (P < .0008-.0283). This observation may explain the predominance of clade B in the community and why antibiotic-resistant hospital-associated E. faecium are often replaced by clade B strains once patients leave the hospital.

The Enterococcus faecalis EbpA Pilus Protein: Attenuation of Expression, Biofilm Formation, and Adherence to Fibrinogen Start with the Rare Initiation Codon ATT.

UNLABELLED: The endocarditis and biofilm-associated pili (Ebp) are important in Enterococcus faecalis pathogenesis, and the pilus tip, EbpA, has been shown to play a major role in pilus biogenesis, biofilm formation, and experimental infections. Based on in silico analyses, we previously predicted that ATT is the EbpA translational start codon, not the ATG codon, 120 bp downstream of ATT, which is annotated as the translational start. ATT is rarely used to initiate protein synthesis, leading to our hypothesis that this codon participates in translational regulation of Ebp production. To investigate this possibility, site-directed mutagenesis was used to introduce consecutive stop codons in place of two lysines at positions 5 and 6 from the ATT, to replace the ATT codon in situ with ATG, and then to revert this ATG to ATT; translational fusions of ebpA to lacZ were also constructed to investigate the effect of these start codons on translation. Our results showed that the annotated ATG does not start translation of EbpA, implicating ATT as the start codon; moreover, the presence of ATT, compared to the engineered ATG, resulted in significantly decreased EbpA surface display, attenuated biofilm, and reduced adherence to fibrinogen. Corroborating these findings, the translational fusion with the native ATT as the initiation codon showed significantly decreased expression of ß-galactosidase compared to the construct with ATG in place of ATT. Thus, these results demonstrate that the rare initiation codon of EbpA negatively regulates EbpA surface display and negatively affects Ebp-associated functions, including biofilm and adherence to fibrinogen. IMPORTANCE: Enterococcus faecalis is among the leading causes of serious infections in the hospital setting, and the endocarditis and biofilm-associated pili (Ebp) have been shown to play significant roles in E. faecalis pathogenesis. Understanding the regulation of virulence is important for the development of new approaches to counteract multidrug-resistant pathogens. We previously predicted that ATT, which has been reported to start protein synthesis only in rare instances, is the most likely translational start codon of EbpA in E. faecalis. Here, we demonstrate that ATT is the initiation codon of EbpA and, relative to a constructed ATG start codon, results in smaller amounts of EbpA on the surface of the cells, attenuating biofilm formation and fibrinogen adherence, phenotypes associated with the ability of E. faecalis to cause infections. This provides the first example of pilus regulation through the use of an ATT initiation codon.

Enterococcus faecium PBP5-S/R, the missing link between PBP5-S and PBP5-R.

During a study to investigate the evolution of ampicillin resistance in Enterococcus faecium, we observed that a number of E. faecium strains, mainly from the recently described subclade A2, showed PBP5 sequences in between PBP5-S and PBP5-R. These hybrid PBP5-S/R patterns reveal a progression of amino acid changes from the S form to the R form of this protein; however, these changes do not strictly correlate with changes in ampicillin MICs.

Contribution of individual Ebp Pilus subunits of Enterococcus faecalis OG1RF to pilus biogenesis, biofilm formation and urinary tract infection.

The endocarditis and biofilm-associated pilus (Ebp) operon is a component of the core genome of Enterococcus faecalis that has been shown to be important for biofilm formation, adherence to host fibrinogen, collagen and platelets, and in experimental endocarditis and urinary tract infection models. Here, we created single and double deletion mutants of the pilus subunits and sortases; next, by combining western blotting, immunoelectron microscopy, and using ebpR in trans to increase pilus production, we identified EbpA as the tip pilin and EbpB as anchor at the pilus base, the latter attached to cell wall by the housekeeping sortase, SrtA. We also confirmed EbpC and Bps as the major pilin and pilin-specific sortase, respectively, both required for pilus polymerization. Interestingly, pilus length was increased and the number of pili decreased by deleting ebpA, while control overexpression of ebpA in trans restored wild-type levels, suggesting a dual role for EbpA in both initiation and termination of pilus polymerization. We next investigated the contribution of each pilin subunit to biofilm formation and UTI. Significant reduction in biofilm formation was observed with deletion of ebpA or ebpC (P<0.001) while ebpB was found to be dispensable; a similar result was seen in kidney CFUs in experimental UTI (ΔebpA, ΔebpC, P≤0.0093; ΔebpB, non-significant, each vs. OG1RF). Hence, our data provide important structural and functional information about these ubiquitous E. faecalis pili and, based on their demonstrated importance in biofilm and infection, suggest EbpA and EbpC as potential targets for antibody-based therapeutic approaches.

Alternative splicing and subfunctionalization generates functional diversity in fungal proteomes.

Alternative splicing is commonly used by the Metazoa to generate more than one protein from a gene. However, such diversification of the proteome by alternative splicing is much rarer in fungi. We describe here an ancient fungal alternative splicing event in which these two proteins are generated from a single alternatively spliced ancestral SKI7/HBS1 gene retained in many species in both the Ascomycota and Basidiomycota. While the ability to express two proteins from a single SKI7/HBS1 gene is conserved in many fungi, the exact mechanism by which they achieve this varies. The alternative splicing was lost in Saccharomyces cerevisiae following the whole-genome duplication event as these two genes subfunctionalized into the present functionally distinct HBS1 and SKI7 genes. When expressed in yeast, the single gene from Lachancea kluyveri generates two functionally distinct proteins. Expression of one of these proteins complements hbs1, but not ski7 mutations, while the other protein complements ski7, but not hbs1. This is the first known case of subfunctionalization by loss of alternative splicing in yeast. By coincidence, the ancestral alternatively spliced gene was also duplicated in Schizosaccharomyces pombe with subsequent subfunctionalization and loss of splicing. Similar subfunctionalization by loss of alternative splicing in fungi also explains the presence of two PTC7 genes in the budding yeast Tetrapisispora blattae, suggesting that this is a common mechanism to preserve duplicate alternatively spliced genes.

First identification of OXA-72 carbapenemase from Acinetobacter pittii in Colombia.

OXA-72 has been reported in few countries around the world. We report the first case in Colombia in an Acinetobacter pittii clinical isolate. The arrival of a new OXA, into a country with high endemic resistance, poses a significant threat, especially because the potential for widespread dissemination is considerable.

Novel VIM metallo-beta-lactamase variant, VIM-24, from a Klebsiella pneumoniae isolate from Colombia.

We report the emergence of a novel VIM variant (VIM-24) in a Klebsiella pneumoniae isolate in Colombia. The isolate displays MICs for carbapenems below the resistance breakpoints, posing a real challenge for its detection. The blaVIM-24 gene was located within a class 1 integron carried on a large plasmid. Further studies are needed to clarify its epidemiological and clinical impact.