Synergistic effects of ceftazidime/avibactam combined with meropenem in a murine model of infection with KPC-producing Klebsiella pneumoniae.

OBJECTIVES: The emergence and expansion of carbapenem-resistant Klebsiella pneumoniae infections is a concern due to the lack of 'first-line' antibiotic treatment options. The ceftazidime/avibactam is an important clinical treatment for carbapenem-resistant K. pneumoniae infections but there is an increasing number of cases of treatment failure and drug resistance. Therefore, a potential solution is combination therapies that result in synergistic activity against K. pneumoniae carbapenemase: producing K. pneumoniae (KPC-Kp) isolates and preventing the emergence of KPC mutants resistant to ceftazidime/avibactam are needed in lieu of novel antibiotics. METHODS: To evaluate their synergistic activity, antibiotic combinations were tested against 26 KPC-Kp strains. Antibiotic resistance profiles, molecular characteristics and virulence genes were investigated by susceptibility testing and whole-genome sequencing. Antibiotic synergy was evaluated by in vitro chequerboard experiments, time-killing curves and dose-response assays. The mouse thigh model was used to confirm antibiotic combination activities in vivo. Additionally, antibiotic combinations were evaluated for their ability to prevent the emergence of ceftazidime/avibactam resistant mutations of blaKPC. RESULTS: The combination of ceftazidime/avibactam plus meropenem showed remarkable synergistic activity against 26 strains and restored susceptibility to both the partnering antibiotics. The significant therapeutic effect of ceftazidime/avibactam combined with meropenem was also confirmed in the mouse model and bacterial loads in the thigh muscle of the combination groups were significantly reduced. Furthermore, ceftazidime/avibactam plus meropenem showed significant activity in preventing the occurrence of resistance mutations. CONCLUSIONS: Our results indicated that the combination of ceftazidime/avibactam plus meropenem offers viable therapeutic alternatives in treating serious infections due to KPC-Kp.

A high-throughput microfluidic nanoimmunoassay for detecting anti-SARS-CoV-2 antibodies in serum or ultralow-volume blood samples.

Novel technologies are needed to facilitate large-scale detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) specific antibodies in human blood samples. Such technologies are essential to support seroprevalence studies and vaccine clinical trials, and to monitor quality and duration of immunity. We developed a microfluidic nanoimmunoassay (NIA) for the detection of anti-SARS-CoV-2 IgG antibodies in 1,024 samples per device. The method achieved a specificity of 100% and a sensitivity of 98% based on the analysis of 289 human serum samples. To eliminate the need for venipuncture, we developed low-cost, ultralow-volume whole blood sampling methods based on two commercial devices and repurposed a blood glucose test strip. The glucose test strip permits the collection, shipment, and analysis of 0.6 µL of whole blood easily obtainable from a simple finger prick. The NIA platform achieves high throughput, high sensitivity, and specificity based on the analysis of 289 human serum samples, and negligible reagent consumption. We furthermore demonstrate the possibility to combine NIA with decentralized and simple approaches to blood sample collection. We expect this technology to be applicable to current and future SARS-CoV-2 related serological studies and to protein biomarker analysis in general.

[Reading an antibiogram in the context of antimicrobial resistance]. / Lecture de l'antibiogramme dans un contexte de résistance aux antibiotiques.

Reading an antibiogram, however simple it may appear at first glance, is full of sometimes implicit but valuable information in the daily practice of the physician. A more subtle reading requires knowledge in the microbiology and pharmacology fields, even more so in the presence of resistant bacteria. The aim of this article is to provide the clinician with some keys to read and understand an antibiogram in the current context.

La lecture d'un antibiogramme, aussi simple qu'elle puisse paraître au premier coup d'œil, regorge d'informations parfois implicites qui peuvent être précieuses dans la pratique quotidienne du médecin clinicien. Une lecture plus approfondie nécessite quelques connaissances de microbiologie et de pharmacologie, ce d'autant plus lorsque la bactérie est multirésistante. Cet article a pour but d'apporter quelques clés de lecture de l'antibiogramme au médecin clinicien dans ce contexte.

Robust T-Cell Responses in Anti-CD20-Treated Patients Following COVID-19 Vaccination: A Prospective Cohort Study.

BACKGROUND: Patients treated with anti-CD20 therapy are particularly at risk of developing severe coronavirus disease 2019 (COVID-19); however, little is known regarding COVID-19 vaccine effectiveness in this population. METHODS: This prospective observational cohort study assesses humoral and T-cell responses after vaccination with 2 doses of mRNA-based COVID-19 vaccines in patients treated with rituximab for rheumatic diseases or ocrelizumab for multiple sclerosis (n = 37), compared to immunocompetent individuals (n = 22). RESULTS: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific antibodies were detectable in only 69.4% of patients and at levels that were significantly lower compared to controls who all seroconverted. In contrast to antibodies, Spike (S)-specific CD4 T cells were equally detected in immunocompetent and anti-CD20 treated patients (85-90%) and mostly of a Th1 phenotype. Response rates of S-specific CD8 T cells were higher in ocrelizumab (96.2%) and rituximab-treated patients (81.8%) as compared to controls (66.7%). S-specific CD4 and CD8 T cells were polyfunctional but expressed more effector molecules in patients than in controls. During follow-up, 3 MS patients without SARS-CoV-2-specific antibody response had a mild breakthrough infection. One of them had no detectable S-specific T cells after vaccination. CONCLUSIONS: Our study suggests that patients on anti-CD20 treatment are able to mount potent T-cell responses to mRNA COVID-19 vaccines, despite impaired humoral responses. This could play an important role in the reduction of complications of severe COVID-19.

Epidemiology and outcomes of bone and joint infections in solid organ transplant recipients.

Bone and joint infection (BJI) epidemiology and outcomes in solid organ transplant recipients (SOTr) remain largely unknown. We aim to describe BJI in a multi-center cohort of SOTr (Swiss Transplant Cohort Study). All consecutive SOTr with BJI (01.05.2008-31.12.2019) were included. A nested case-control study to identify risk factors for BJI was performed. Among 4482 patients, 61 SOTr with 82 BJI were included, at an incidence of 1.4% (95% CI 1.1-1.7), higher in heart and kidney-pancreas SOTr (Gray's test p < .01). Although BJI were predominately late events (median of 18.5 months post-SOT), most infections occurred during the first year post-transplant in thoracic SOTr. Diabetic foot osteomyelitis was the most frequent infection (38/82, 46.3%), followed by non-vertebral osteomyelitis (26/82, 31.7%). Pathogens included Gram-positive cocci (70/131, 53.4%), Gram-negative bacilli (34/131, 26.0%), and fungi (9/131, 6.9%). BJI predictors included male gender (OR 2.94, 95% CI 1.26-6.89) and diabetes (OR 2.97, 95% CI 1.34-6.56). Treatment failure was observed in 25.9% (21/81) patients and 1-year mortality post-BJI diagnosis was 14.8% (9/61). BJI remain a rare event in SOTr, associated with subtle clinical presentations, high morbidity and relapses, requiring additional studies in the future.

Autoantibodies against apolipoprotein A-1 after COVID-19 predict symptoms persistence.

BACKGROUND: SARS-CoV-2 infection triggers different auto-antibodies, including anti-apolipoprotein A-1 IgGs (AAA1), which could be of concern as mediators of persistent symptoms. We determined the kinetics of AAA1 response over after COVID-19 and the impact of AAA1 on the inflammatory response and symptoms persistence. METHODS: All serologies were assessed at one, three, six and twelve months in 193 hospital employees with COVID-19. ROC curve analyses and logistic regression models (LRM) were used to determine the prognostic accuracy of AAA1 and their association with patient-reported COVID-19 symptoms persistence at 12 months. Interferon (IFN)-α and-γ production by AAA1-stimulated human monocyte-derived macrophages (HMDM) was assessed in vitro. RESULTS: AAA1 seropositivity was 93% at one month and declined to 15% at 12 months after COVID-19. Persistent symptoms at 12 months were observed in 45.1% of participants, with a predominance of neurological (28.5%), followed by general (15%) and respiratory symptoms (9.3%). Over time, strength of correlations between AAA1 and anti-SARS-COV2 serologies decreased, but remained significant. From the 3rd month on, AAA1 levels predicted persistent respiratory symptoms (area under the curves 0.72-0.74; p < 0.001), independently of disease severity, age and gender (adjusted odds ratios 4.81-4.94; p = 0.02), while anti-SARS-CoV-2 serologies did not. AAA1 increased IFN-α production by HMDMs (p = 0.03), without affecting the IFN-γ response. CONCLUSION: COVID-19 induces a marked though transient AAA1 response, independently predicting one-year persistence of respiratory symptoms. By increasing IFN-α response, AAA1 may contribute to persistent symptoms. If and how AAA1 levels assessment could be of use for COVID-19 risk stratification remains to be determined.

Insights into the global effect on Staphylococcus aureus growth arrest by induction of the endoribonuclease MazF toxin.

A crucial bacterial strategy to avoid killing by antibiotics is to enter a growth arrested state, yet the molecular mechanisms behind this process remain elusive. The conditional overexpression of mazF, the endoribonuclease toxin of the MazEF toxin-antitoxin system in Staphylococcus aureus, is one approach to induce bacterial growth arrest, but its targets remain largely unknown. We used overexpression of mazF and high-throughput sequence analysis following the exact mapping of non-phosphorylated transcriptome ends (nEMOTE) technique to reveal in vivo toxin cleavage sites on a global scale. We obtained a catalogue of MazF cleavage sites and unearthed an extended MazF cleavage specificity that goes beyond the previously reported one. We correlated transcript cleavage and abundance in a global transcriptomic profiling during mazF overexpression. We observed that MazF affects RNA molecules involved in ribosome biogenesis, cell wall synthesis, cell division and RNA turnover and thus deliver a plausible explanation for how mazF overexpression induces stasis. We hypothesize that autoregulation of MazF occurs by directly modulating the MazEF operon, such as the rsbUVW genes that regulate the sigma factor SigB, including an observed cleavage site on the MazF mRNA that would ultimately play a role in entry and exit from bacterial stasis.

Prevalence of Immunoglobulin G (IgG) Against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and Evaluation of a Rapid MEDsan IgG Test in Children Seeking Medical Care.

In 208 children seeking medical care, the seropositivity rate of anti-SARS-CoV-2 IgG antibodies was 8.7%, suggesting an infection rate similar to that observed in adults but >100-fold the incidence of RT-PCR-confirmed pediatric cases. Compared with the gold-standard combined ELISA + immunofluorescence, the MEDsan IgG rapid diagnostic test performed accurately.

An Emerging Clone, Klebsiellapneumoniae Carbapenemase 2-Producing K. pneumoniae Sequence Type 16, Associated With High Mortality Rates in a CC258-Endemic Setting.

BACKGROUND: Carbapenemase-producing Klebsiella pneumoniae has become a global priority, not least in low- and middle-income countries. Here, we report the emergence and clinical impact of a novel Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-KP) sequence type (ST) 16 clone in a clonal complex (CC) 258-endemic setting. METHODS: In a teaching Brazilian hospital, a retrospective cohort of adult KPC-KP bloodstream infection (BSI) cases (January 2014 to December 2016) was established to study the molecular epidemiology and its impact on outcome (30-day all-cause mortality). KPC-KP isolates underwent multilocus sequence typing. Survival analysis between ST/CC groups and risk factors for fatal outcome (logistic regression) were evaluated. Representative isolates underwent whole-genome sequencing and had their virulence tested in a Galleria larvae model. RESULTS: One hundred sixty-five unique KPC-KP BSI cases were identified. CC258 was predominant (66%), followed by ST16 (12%). The overall 30-day mortality rate was 60%; in contrast, 95% of ST16 cases were fatal. Patients' severity scores were high and baseline clinical variables were not statistically different across STs. In multivariate analysis, ST16 (odds ratio [OR], 21.4; 95% confidence interval [CI], 2.3-202.8; P = .008) and septic shock (OR, 11.9; 95% CI, 4.2-34.1; P < .001) were independent risk factors for fatal outcome. The ST16 clone carried up to 14 resistance genes, including blaKPC-2 in an IncFIBpQIL plasmid, KL51 capsule, and yersiniabactin virulence determinants. The ST16 clone was highly pathogenic in the larvae model. CONCLUSIONS: Mortality rates were high in this KPC-KP BSI cohort, where CC258 is endemic. An emerging ST16 clone was associated with high mortality. Our results suggest that even in endemic settings, highly virulent clones can rapidly emerge demanding constant monitoring.

Diagnostic accuracy of Augurix COVID-19 IgG serology rapid test.

AIMS: To validate the diagnostic accuracy of the Augurix SARS-CoV-2 IgM/IgG rapid immunoassay diagnostic test (RDT) for COVID-19. METHODS: In this unmatched 1:1 case-control study, blood samples from 46 real-time RT-PCR-confirmed SARS-CoV-2 hospitalized cases and 45 healthy donors (negative controls) were studied. Diagnostic accuracy of the IgG RDT was assessed against both an in-house recombinant spike-expressing immunofluorescence assay (rIFA), as an established reference method (primary endpoint), and the Euroimmun SARS-CoV-2 IgG enzyme-linked immunosorbent assays (ELISA) (secondary endpoint). RESULTS: COVID-19 patients were more likely to be male (61% vs 20%; P = .0001) and older (median 66 vs 47 years old; P < .001) than controls. Whole blood IgG-RDT results showed 86% and 93% overall Kendall concordance with rIFA and IgG ELISA, respectively. IgG RDT performances were similar between plasma and whole blood. Overall, RDT sensitivity was 88% (95% confidence interval [95%CI]: 70-96), specificity 98% (95%CI: 90-100), PPV 97% (95%CI: 80-100) and NPV 94% (95%CI: 84-98). The IgG-RDT carried out from 0 to 6 days, 7 to 14 days and > 14 days after the SARS-CoV-2 RT-PCR test displayed 30%, 73% and 100% positivity rates in the COVID-19 group, respectively. When considering samples taken >14 days after RT-PCR diagnosis, NPV was 100% (95%CI:90-100), and PPV was 100% (95%CI:72-100). CONCLUSIONS: The Augurix IgG-RDT done in whole blood displays a high diagnostic accuracy for SARS-CoV-2 IgG in high COVID-19 prevalence settings, where its use could be considered in the absence of routine diagnostic serology facilities.

Outbreak of Hypervirulent Multidrug-resistant Klebsiella variicola Causing High Mortality in Neonates in Bangladesh.

We report a clonal outbreak of multidrug-resistant (MDR) Klebsiella variicola (sequence type [ST] 771) in a Bangladeshi neonatal unit from October 2016 to January 2017, associated with high mortality (54.5%). During the outbreak, K. variicola ST771 acquired an MDR plasmid harboring blaNDM-1, linked to high exposure to ceftriaxone and amikacin.

Pancreas preservation fluid microbial contamination is associated with poor islet isolation outcomes - a multi-centre cohort study.

The microbiological safety of islet preparations is paramount. Preservation medium contamination is frequent, and its impact on islet yield and function remains unclear. Microbiological samples collected during islet isolations from 2006 to 2016 were analyzed and correlated to isolation and allo- and autotransplantation outcomes. Microbial contamination of preservation medium was found in 64.4% of processed donor pancreases (291/452). We identified 464 microorganisms including Staphylococcus (253/464, 54.5%), Streptococcus (31/464, 6.7%), and Candida species (25/464, 5.4%). Microbial contamination was associated with longer warm and cold ischemia times and lower numbers of postpurification islet equivalents, purity, transplant rate, and stimulation index (all P < 0.05). Six percent of the preparations accepted for transplantation showed microbial contamination after isolation (12/200); 9 of 12 were Candida species. Six patients were transplanted with a sample with late microbial growth discovered after the infusion. Insulin independence rate was not affected. This risk of transplanting a contaminated islets preparation was reduced by half following the implementation of an additional sampling after 24 h of islet culture. Pancreas preservation fluid microbial contamination is associated with lower transplant rate and poorer in vitro function, but not with changes in graft survival. Culture medium testing 1 day after isolation reduces the risk of incidental transplantation with contaminated islets.

Rifampin Resistance rpoB Alleles or Multicopy Thioredoxin/Thioredoxin Reductase Suppresses the Lethality of Disruption of the Global Stress Regulator spx in Staphylococcus aureus.

UNLABELLED: Staphylococcus aureus is capable of causing a remarkable spectrum of disease, ranging from mild skin eruptions to life-threatening infections. The survival and pathogenic potential of S. aureus depend partly on its ability to sense and respond to changes in its environment. Spx is a thiol/oxidative stress sensor that interacts with the C-terminal domain of the RNA polymerase RpoA subunit, leading to changes in gene expression that help sustain viability under various conditions. Using genetic and deep-sequencing methods, we show that spx is essential in S. aureus and that a previously reported Δspx strain harbored suppressor mutations that allowed it to grow without spx One of these mutations is a single missense mutation in rpoB (a P-to-L change at position 519 encoded by rpoB [rpoB-P519L]) that conferred high-level resistance to rifampin. This mutation alone was found to be sufficient to bypass the requirement for spx The generation of rifampin resistance libraries led to the discovery of an additional rpoB mutation, R484H, which supported strains with the spx disruption. Other rifampin resistance mutations either failed to support the Δspx mutant or were recovered at unexpectedly low frequencies in genetic transduction experiments. The amino acid residues encoded by rpoB-P519L and -R484H map in close spatial proximity and comprise a highly conserved region of RpoB. We also discovered that multicopy expression of either trxA (encoding thioredoxin) or trxB (encoding thioredoxin reductase) supports strains with the deletion of spx Our results reveal intriguing properties, especially of RNA polymerase, that compensate for the loss of an essential gene that is a key mediator of diverse processes in S. aureus, including redox and thiol homeostasis, antibiotic resistance, growth, and metabolism. IMPORTANCE: The survival and pathogenicity of S. aureus depend on complex genetic programs. An objective for combating this insidious organism entails dissecting genetic regulatory circuits and discovering promising new targets for therapeutic intervention. In this study, we discovered that Spx, an RNA polymerase-interacting stress regulator implicated in many stress responses in S. aureus, including responses to oxidative and cell wall antibiotics, is essential. We describe two mechanisms that suppress the lethality of spx disruption. One mechanism highlights how only certain rifampin resistance-encoding alleles of RpoB confer new properties on RNA polymerase, with important mechanistic implications. We describe additional stress conditions where the loss of spx is deleterious, thereby highlighting Spx as a multifaceted regulator and attractive drug discovery target.

Carriage of third-generation cephalosporin-resistant and carbapenem-resistant Enterobacterales among children in sub-Saharan Africa: a systematic review and meta-analysis.

Background: The increasing resistance of Enterobacterales to third-generation cephalosporins and carbapenems in sub-Saharan Africa (SSA) is a major public health concern. We did a systematic review and meta-analysis of studies to estimate the carriage prevalence of Enterobacterales not susceptible to third-generation cephalosporins or carbapenems among paediatric populations in SSA. Methods: We performed a systematic literature review and meta-analysis of cross-sectional and cohort studies to estimate the prevalence of childhood (0-18 years old) carriage of extended-spectrum cephalosporin-resistant Enterobacterales (ESCR-E) or carbapenem-resistant Enterobacterales (CRE) in SSA. Medline, EMBASE and the Cochrane Library were searched for studies published from 1 January 2005 to 1 June 2022. Studies with <10 occurrences per bacteria, case reports, and meta-analyses were excluded. Quality and risk of bias were assessed using the Newcastle-Ottawa scale. Meta-analyses of prevalences and odds ratios were calculated using generalised linear mixed-effects models. Heterogeneity was assessed using I2 statistics. The protocol is available on PROSPERO (CRD42021260157). Findings: Of 1111 studies examined, 40 met our inclusion criteria, reporting on the carriage prevalence of Enterobacterales in 9408 children. The pooled carriage prevalence of ESCR-E was 32.2% (95% CI: 25.2%-40.2%). Between-study heterogeneity was high (I2 = 96%). The main sources of bias pertained to participant selection and the heterogeneity of the microbiological specimens. Carriage proportions were higher among sick children than healthy ones (35.7% vs 16.9%). The pooled proportion of nosocomial acquisition was 53.8% (95% CI: 32.1%-74.1%) among the 922 children without ESCR-E carriage at hospital admission. The pooled odds ratio of ESCR-E carriage after antibiotic treatment within the previous 3 months was 3.20 (95% CI: 2.10-4.88). The proportion of pooled carbapenem-resistant for Enterobacterales was 3.6% (95% CI: 0.7%-16.4%). Interpretation: This study suggests that ESCR-E carriage among children in SSA is frequent. Microbiology capacity and infection control must be scaled-up to reduce the spread of those multidrug-resistant microorganisms. Funding: There was no funding source for this study.

Antimicrobial resistance in Enterobacterales infections among children in sub-Saharan Africa: a systematic review and meta-analysis.

Background: The burden of antimicrobial resistance (AMR) has been estimated to be the highest in sub-Saharan Africa (SSA). The current study estimated the proportion of drug-resistant Enterobacterales causing infections in SSA children. Methods: We searched MEDLINE/PubMed, Embase and the Cochrane Library to identify retrospective and prospective studies published from 01/01/2005 to 01/06/2022 reporting AMR of Enterobacterales causing infections in sub-Saharan children (0-18 years old). Studies were excluded if they had unclear documentation of antimicrobial susceptibility testing methods or fewer than ten observations per bacteria. Data extraction and quality appraisal were conducted by two authors independently. The primary outcome was the proportion of Enterobacterales resistant to antibiotics commonly used in paediatrics. Proportions were combined across studies using mixed-effects logistic regression models per bacteria and per antibiotic. Between-study heterogeneity was assessed using the I2 statistic. The protocol was registered with PROSPERO (CRD42021260157). Findings: After screening 1111 records, 122 relevant studies were included, providing data on more than 30,000 blood, urine and stool isolates. Escherichia coli and Klebsiella spp. were the predominant species, both presenting high proportions of resistance to third-generation cephalosporins, especially in blood cultures: 40.6% (95% CI: 27.7%-55%; I2: 85.7%, number of isolates (n): 1032) and 84.9% (72.8%-92.2%; I2: 94.1%, n: 2067), respectively. High proportions of resistance to other commonly used antibiotics were also observed. E. coli had high proportions of resistance, especially for ampicillin (92.5%; 95% CI: 76.4%-97.9%; I2: 89.8%, n: 888) and gentamicin (42.7%; 95% CI: 30%-56.5%; I2: 71.9%, n: 968). Gentamicin-resistant Klebsiella spp. were also frequently reported (77.6%; 95% CI: 65.5%-86.3%; I2: 91.6%, n: 1886). Interpretation: High proportions of resistance to antibiotics commonly used for empirical treatment of infectious syndromes were found for Enterobacterales in sub-Saharan children. There is a critical need to better identify local patterns of AMR to inform and update clinical guidelines for better treatment outcomes. Funding: No funding was received.

Antibiotic heteroresistance in ESKAPE pathogens, from bench to bedside.

BACKGROUND: Heteroresistance refers to subpopulation-mediated differential antimicrobial susceptibility within a clonal bacterial population. Usually, it designates a resistant subpopulation identified within an isolate considered susceptible by classical antimicrobial susceptibility testing. Heteroresistance lacks a uniform microbiological definition for diagnostic laboratories, and its clinical impact remains unclear for most bacterial species. OBJECTIVES: This narrative review aims to provide a practical overview on the latest developments in the field of heteroresistance for both clinical microbiologists and physicians, with a particular focus on ESKAPE pathogens. SOURCES: A literature search was performed on Pubmed and Google with the key words heteroresistance (heterogeneity OR heterogeneous) AND antibiotic resistance. Among the 836 publications selected based on their abstracts, the most relevant for the detection, epidemiology and clinical impact of heteroresistance in ESKAPE pathogens are discussed here. CONTENT: Heteroresistance is only clearly defined for heterogeneous vancomycin intermediate Staphylococcus aureus. We compiled a larger microbiological definition to be applicable to other bacterial species and antibiotics in the clinical context. We highlighted the key technical points of population analysis profile, which is the reference standard for detecting heteroresistance. Heteroresistance to polymyxins, ß-lactams (carbapenems, cefiderocol), fosfomycin, tigecycline and aminoglycosides is frequently reported in multidrug-resistant gram-negative pathogens. Treatment failure due to heteroresistance has been described in case reports or retrospective studies, so far confirmed by meta-analyses in the case of heterogeneous vancomycin intermediate S. aureus only. Finally, to treat pandrug-resistant bacterial infections, the option of targeting susceptible subpopulations of resistant isolates using tailored antibiotic combinations is also discussed. IMPLICATIONS: Systematic heteroresistance screening by clinical laboratories is not currently recommended. Nevertheless, we should be aware of this phenomenon, and in specific cases, such as treatment failure, heteroresistance should be tested by reference laboratories. Additional studies using standardized methods are needed to improve our understanding of heteroresistance and further assess its clinical impact.

Plasmid-mediated fosfomycin resistance in Escherichia coli isolates of worldwide origin.

OBJECTIVES: Fosfomycin is a first-line treatment for uncomplicated urinary tract infections (UTIs) in several European countries, and it is increasingly becoming the treatment of choice globally. Resistance to fosfomycin in Escherichia coli can be exerted through several mechanisms, including the acquisition of fosfomycin-modifying enzymes, of which the FosA-type enzymes are the most common. This study analysed, both phenotypically and genotypically, an international collection of E. coli strains harbouring acquired fosA genes. METHODS: Thirty-one fosA-positive E. coli isolates were obtained from both clinical and environmental sources, from seven countries (Portugal (n = 12), Switzerland (n = 9), China (n = 3), France (n = 2), Nepal (n = 2), South Africa (n = 2), Kuwait (n = 1)). MICs were determined according to EUCAST guidelines. Whole genome sequencing (WGS) was performed on 23 isolates, and complete fosA plasmid sequences were determined for 12. Conjugation assays were performed on seven isolates. RESULTS: All isolates exhibited high-level resistance to fosfomycin (64 to >256 mg/L). WGS of 23 isolates identified 17 sequence types (STs), and 16 harboured fosA3, four fosA4, two fosA8, and one fosA10. ESBLs, pAmpC, or carbapenemase genes were present in 15, four, and three isolates, respectively. The fosA plasmids of 12 isolates were determined and were diverse in size (∼67 kb to ∼235 kb), resistance gene carriage, and replicon types. Six fosA plasmids additionally carried ESBL or carbapenemase genes. Conjugation assays, performed on seven isolates harbouring diverse plasmids, identified that all were capable of being transmitted. CONCLUSION: This study highlights the necessity of the surveillance and close monitoring of fosfomycin resistance in E. coli, essential to maintain the optimal use of this treatment option.

The posttranslocational chaperone lipoprotein PrsA is involved in both glycopeptide and oxacillin resistance in Staphylococcus aureus.

Understanding in detail the factors which permit Staphylococcus aureus to counteract cell wall-active antibiotics is a prerequisite to elaborating effective strategies to prolong the usefulness of these drugs and define new targets for pharmacological intervention. Methicillin-resistant S. aureus (MRSA) strains are major pathogens of hospital-acquired and community-acquired infections and are most often treated with glycopeptides (vancomycin and teicoplanin) because of their resistance to most penicillins and a limited arsenal of clinically proven alternatives. In this study, we examined PrsA, a lipid-anchored protein of the parvulin PPIase family (peptidyl-prolyl cis/trans isomerase) found ubiquitously in all Gram-positive species, in which it assists posttranslocational folding at the outer surface of the cytoplasmic membrane. We show by both genetic and biochemical assays that prsA is directly regulated by the VraRS two-component sentinel system of cell wall stress. Disruption of prsA is tolerated by S. aureus, and its loss results in no detectable overt macroscopic changes in cell wall architecture or growth rate under nonstressed growth conditions. Disruption of prsA leads, however, to notable alterations in the sensitivity to glycopeptides and dramatically decreases the resistance of COL (MRSA) to oxacillin. Quantitative transcriptional analysis reveals that prsA and vraR are coordinately upregulated in a panel of stable laboratory and clinical glycopeptide-intermediate S. aureus (GISA) strains compared to their susceptible parents. Collectively, our results point to a role for prsA as a facultative facilitator of protein secretion or extracellular folding and provide a framework for understanding why prsA is a key element of the VraRS-mediated cell wall stress response.

Characterization of Amino Acid Substitution W20S in MgrB Involved in Polymyxin Resistance in Klebsiella pneumoniae.

In the major human pathogen Klebsiella pneumoniae, MgrB inactivation by disruptive insertion sequence (IS) elements and mutations leading to early termination are known to play an important role in polymyxin resistance. In this study, we examined a collection of invasive blaKPC-2-producing K. pneumoniae isolates belonging to the high-risk clone sequence type 258 (ST258) displaying high rates of resistance to many antimicrobials, including polymyxins. We identified a deleterious substitution (W20S) in MgrB and confirmed by genetic complementation analysis that this variant was inactive, leading to increased polymyxin B and colistin MICs. IMPORTANCE Carbapenem-resistant Gram-negative bacteria are designated critical pathogens by the World Health Organization. Polymyxins (i.e., polymyxin B and colistin) are last-resort antibiotics and particularly useful against these multidrug-resistant bacteria. In Klebsiella pneumoniae, the inactivation of MgrB, a negative regulator of PhoPQ, was shown to be the major pathway leading to colistin resistance. While gene disruption by insertion sequence (IS) elements and mutations leading to early termination (stop codons) are frequent, deleterious mutations are not observed frequently and have not been characterized. Here, we identified a deleterious substitution (W20S) in MgrB among a collection of bloodstream infection, blaKPC-2-producing K. pneumoniae sequence type 258 (ST258) isolates, displaying high rates of resistance to polymyxins and associated with a high mortality rate. The dissemination of such a MgrB-W20S mutation leading to polymyxin resistance within the ST258 high-risk clone background is problematic and thus warrants particular attention.