Resistance development in Escherichia coli to delafloxacin at pHs 6.0 and 7.3 compared to ciprofloxacin.

Understanding the resistance mechanisms of antibiotics in the micro-environment of the infection is important to assess their clinical applicability and potentially prevent resistance development. We compared the laboratory resistance evolution of Escherichia coli to delafloxacin (DLX) compared to ciprofloxacin (CIP), the co-resistance evolution, and underlying resistance mechanisms at different pHs. Three clones from each of the eight clinical E. coli isolates were subjected to subinhibitory concentrations of DLX or CIP in parallel at either pH 7.3 or 6.0. Minimum inhibitory concentrations (MICs) were regularly tested (at respective pHs), and the antibiotic concentration was adjusted accordingly. After 30 passages, MICs were determined in the presence of the efflux pump inhibitor phenylalanine-arginine-ß-naphthylamide. Whole genome sequencing of the parental isolates and their resistant derivatives (n = 54) was performed. Complementation assays were carried out for selected mutations. Quantitative PCR and efflux experiments were carried out for selected derivatives. For DLX-challenged strains, resistance to DLX evolved much slower in acidic than in neutral pH, whereas for CIP-challenged strains, the opposite was the case. Mutations in the quinolone resistance-determining region were mainly seen in CIP-challenged E. coli, whereas a multifactorial mechanism including mutations in efflux-related genes played a role in DLX resistance evolution (predominantly at pH 6.0). This work provides novel insights into the resistance mechanisms of E. coli to delafloxacin and highlights the importance of understanding micro-environmental conditions at the infection site that might affect the true clinical efficacy of antibiotics and challenges our current antibiotic susceptibility-testing paradigm.

Cefepime Population Pharmacokinetics, Antibacterial Target Attainment, and Estimated Probability of Neurotoxicity in Critically Ill Patients.

Cefepime has been reported to cause concentration-related neurotoxicity, especially in critically ill patients with renal failure. This evaluation aimed to identify a dosing regimen providing a sufficient probability of target attainment (PTA) and the lowest justifiable risk of neurotoxicity in critically ill patients. A population pharmacokinetic model was developed based on plasma concentrations over four consecutive days obtained from 14 intensive care unit (ICU) patients. The patients received a median dose of 2,000 mg cefepime by 30-min intravenous infusions with dosing intervals of every 8 h (q8h) to q24h. A time that the free drug concentration exceeds the MIC over the dosing interval (fT>MIC) of 65% and an fT>2×MIC of 100% were defined as treatment targets. Monte Carlo simulations were carried out to identify a dosing regimen for a PTA of 90% and a probability of neurotoxicity not exceeding 20%. A two-compartment model with linear elimination best described the data. Estimated creatinine clearance was significantly related to the clearance of cefepime in nondialysis patients. Interoccasion variability on clearance improved the model, reflecting dynamic clearance changes. The evaluations suggested combining thrice-daily administration as an appropriate choice. In patients with normal renal function (creatinine clearance, 120 mL/min), for the pharmacodynamics target of 100% fT>2×MIC and a PTA of 90%, a dose of 1,333 mg q8h was found to be related to a probability of neurotoxicity of ≤20% and to cover MICs up to 2 mg/L. Continuous infusion appears to be superior to other dosing regimens by providing higher efficacy and a low risk of neurotoxicity. The model makes it possible to improve the predicted balance between cefepime efficacy and neurotoxicity in critically ill patients. (This study has been registered at ClinicalTrials.gov under registration no. NCT01793012).

Clinical symptoms of SARS-CoV-2 breakthrough infection during the Omicron period in relation to baseline immune status and booster vaccination-A prospective multicentre cohort of health professionals (SURPRISE study).

The effects of different types of pre-existing immunity on the frequency of clinical symptoms caused by the SARS-CoV-2 breakthrough infection were prospectively assessed in healthcare workers during the Omicron period. Among 518 participants, hybrid immunity was associated with symptom reduction for dizziness, muscle or limb pain and headache as compared to vaccination only. Moreover, the frequencies of dizziness, cough and muscle or limb pain were lower in participants who had received a booster vaccine dose. Thus, hybrid immunity appeared to be superior in preventing specific symptoms during breakthrough infection compared to vaccination alone. A booster vaccine dose conferred additional symptom reduction.

Multifactorial resistance mechanisms associated with resistance to ceftazidime-avibactam in clinical Pseudomonas aeruginosa isolates from Switzerland.

Background: Increasing reports of multidrug resistance (MDR) in clinical Pseudomonas aeruginosa have led to a necessity for new antimicrobials. Ceftazidime-avibactam (CZA) is indicated for use against MDR P. aeruginosa across a broad range of infection types and particularly those that are carbapenem resistant. This study sought to determine the molecular mechanisms of CZA and imipenem (IPM)-resistance in clinical P. aeruginosa isolates obtained from Swiss hospitals. Methods: Clinical P. aeruginosa isolates were obtained from inpatients in three hospitals in Switzerland. Susceptibility was determined by either antibiotic disc testing or broth microdilution according to EUCAST methodology. AmpC activity was determined using cloxacillin and efflux activity was determined using phenylalanine-arginine ß-naphthylamide, in agar plates. Whole Genome Sequencing was performed on 18 clinical isolates. Sequence types (STs) and resistance genes were ascertained using the Centre for Genomic Epidemiology platform. Genes of interest were extracted from sequenced isolates and compared to reference strain P. aeruginosa PAO1. Results: Sixteen different STs were identified amongst the 18 isolates in this study indicating a high degree of genomic diversity. No carbapenemases were detected but one isolate did harbor the ESBL bla PER-1. Eight isolates were CZA-resistant with MICs ranging from 16-64 mg/L, and the remaining ten isolates had either low/wildtype MICs (n=6; 1-2 mg/L) or elevated, but still susceptible, MICs (n=4; 4-8 mg/L). Ten isolates were IPM-resistant, seven of which had mutations resulting in truncations of OprD, and the remaining nine IPM-susceptible isolates had intact oprD genes. Within CZA-R isolates, and those with reduced susceptibility, mutations resulting in ampC derepression, OprD loss, mexAB overexpression and ESBL (bla PER-1) carriage were observed in various combinations and one harbored a truncation of the PBP4 dacB gene. Within the six isolates with wildtype-resistance levels, five had no mutations that would affect any antimicrobial resistance (AMR) genes of interest when compared to PAO1. Conclusion: This preliminary study highlights that CZA-resistance in P. aeruginosa is multifactorial and could be caused by the interplay between different resistance mechanisms including ESBL carriage, increased efflux, loss of permeability and derepression of its intrinsic ampC.

Brief Report: Does Menopause Transition Influence Viral Suppression and Adherence in Women Living With HIV?

BACKGROUND: Increasing numbers of women living with HIV transition through menopause. It is unclear whether this transition has an impact on treatment adherence, viral suppression, psychiatric comorbidities, or drug use. We aimed at examining adherence and viral suppression during the perimenopausal period and explored the influence of psychiatric comorbidities and active injection drug use (IDU). SETTING: Retrospective Swiss HIV Cohort Study analysis from January 2010 to December 2018. METHODS: We explored perimenopausal and postmenopausal trends of viral blips, low-level viremia, viral failure, adherence, psychiatric comorbidities, and IDU using interrupted time series models. RESULTS: Rates of depression and psychiatric care increased during perimenopause before decreasing afterward. Negative treatment outcomes such as viral blips, low-level viremia, viral failure, and low adherence steadily declined while transitioning through menopause-this was also true for subgroups of women with depression, psychiatric treatment, and active IDU. CONCLUSIONS: Increased rates of depression and psychiatric care while transitioning through menopause do not result in lower rates of adherence or viral suppression in women living with HIV in Switzerland.

Risk and symptoms of COVID-19 in health professionals according to baseline immune status and booster vaccination during the Delta and Omicron waves in Switzerland-A multicentre cohort study.

BACKGROUND: Knowledge about protection conferred by previous Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection and/or vaccination against emerging viral variants allows clinicians, epidemiologists, and health authorities to predict and reduce the future Coronavirus Disease 2019 (COVID-19) burden. We investigated the risk and symptoms of SARS-CoV-2 (re)infection and vaccine breakthrough infection during the Delta and Omicron waves, depending on baseline immune status and subsequent vaccinations. METHODS AND FINDINGS: In this prospective, multicentre cohort performed between August 2020 and March 2022, we recruited hospital employees from ten acute/nonacute healthcare networks in Eastern/Northern Switzerland. We determined immune status in September 2021 based on serology and previous SARS-CoV-2 infections/vaccinations: Group N (no immunity); Group V (twice vaccinated, uninfected); Group I (infected, unvaccinated); Group H (hybrid: infected and ≥1 vaccination). Date and symptoms of (re)infections and subsequent (booster) vaccinations were recorded until March 2022. We compared the time to positive SARS-CoV-2 swab and number of symptoms according to immune status, viral variant (i.e., Delta-dominant before December 27, 2021; Omicron-dominant on/after this date), and subsequent vaccinations, adjusting for exposure/behavior variables. Among 2,595 participants (median follow-up 171 days), we observed 764 (29%) (re)infections, thereof 591 during the Omicron period. Compared to group N, the hazard ratio (HR) for (re)infection was 0.33 (95% confidence interval [CI] 0.22 to 0.50, p < 0.001) for V, 0.25 (95% CI 0.11 to 0.57, p = 0.001) for I, and 0.04 (95% CI 0.02 to 0.10, p < 0.001) for H in the Delta period. HRs substantially increased during the Omicron period for all groups; in multivariable analyses, only belonging to group H was associated with protection (adjusted HR [aHR] 0.52, 95% CI 0.35 to 0.77, p = 0.001); booster vaccination was associated with reduction of breakthrough infection risk in groups V (aHR 0.68, 95% CI 0.54 to 0.85, p = 0.001) and H (aHR 0.67, 95% CI 0.45 to 1.00, p = 0.048), largely observed in the early Omicron period. Group H (versus N, risk ratio (RR) 0.80, 95% CI 0.66 to 0.97, p = 0.021) and participants with booster vaccination (versus nonboosted, RR 0.79, 95% CI 0.71 to 0.88, p < 0.001) reported less symptoms during infection. Important limitations are that SARS-CoV-2 swab results were self-reported and that results on viral variants were inferred from the predominating strain circulating in the community at that time, rather than sequencing. CONCLUSIONS: Our data suggest that hybrid immunity and booster vaccination are associated with a reduced risk and reduced symptom number of SARS-CoV-2 infection during Delta- and Omicron-dominant periods. For previously noninfected individuals, booster vaccination might reduce the risk of symptomatic Omicron infection, although this benefit seems to wane over time.

It is complicated: Potential short- and long-term impact of coronavirus disease 2019 (COVID-19) on antimicrobial resistance-An expert review.

As of December 2021, the coronavirus disease 2019 (COVID-19) pandemic has claimed millions of deaths and caused disruptions in health systems around the world. The short- and long-term effects of COVID-19 on antimicrobial resistance (AMR), which was already a global threat before the pandemic, are manifold and complex. In this expert review, we summarize how COVID-19 might be affecting AMR in the short term (by influencing the key determinants antibiotic use, infection control practices and international/local mobility) and which additional factors might play a role in the long term. Whereas reduced outpatient antibiotic use in high-income countries, increased awareness for hand hygiene, and reduced mobility have likely mitigated the emergence and spread of AMR in the short term, factors such as overuse of antibiotics in COVID-19 patients, shortage of personal protective equipment, lack of qualified healthcare staff, and patient overcrowding have presumably facilitated its propagation. Unsurprisingly, international and national AMR surveillance data for 2020 show ambiguous trends. Although disruptions in antibiotic stewardship programs, AMR surveillance and research might promote the spread of AMR, other developments could prove beneficial to the cause in the long term. These factors include the increased public awareness for infectious diseases and infection control issues, the strengthening of the One Health perspective as outlined by the Centers for Disease Control and Prevention, and the unprecedented number of international research collaborations and platforms. These factors could even serve as leverage and provide opportunities to better combat AMR in the future.

Women with HIV transitioning through menopause: Insights from the Swiss HIV Cohort Study (SHCS).

OBJECTIVES: We aimed to assess prevalence and age at menopause, identify factors associated with early menopause and explore the provision and utilization of healthcare in women living with HIV in Switzerland. METHODS: This was a retrospective Swiss HIV Cohort Study analysis from January 2010 to December 2018. Descriptive statistics to characterise the population and menopause onset. Logistic regression analysis to identify risk factors for early menopause. RESULTS: Of all women in the SHCS, the proportion of postmenopausal women tripled from 11.5% (n = 274) in 2010 to 36.1% (n = 961) in 2018. The median age at menopause was 50 years. Early menopause (< 45 years) occurred in 115 (10.2%) women and premature ovarian insufficiency (POI) (< 40 years) in 23 (2%) women. Early menopause was associated with black ethnicity (52.2% vs. 21.6%, p < 0.001), but not with HIV acquisition mode, CDC stage, viral suppression, CD4 cell count, hepatitis C, smoking or active drug use. While 92% of the postmenopausal women underwent a gynaecological examination during the 36 months before menopause documentation, only 27% received a bone mineral density measurement within 36 months after the last bleed and 11% were on hormone replacement therapy at the time of menopause documentation. CONCLUSIONS: The median age of women living with HIV at menopause is around 2 years lower than that reported for HIV-negative women in Switzerland. HIV care providers need to adapt their services to the requirements of the increasing number of women living with HIV transitioning through menopause. They should be able to recognize menopause-associated symptoms and improve access to bone mineral density measurement as well as hormone replacement therapy.

AmpC hyperproduction in a Cedecea davisae implant-associated bone infection during treatment: a case report and therapeutic implications.

BACKGROUND: Data on antimicrobial resistance mechanisms are scanty for Cedecea spp., with very variable antibiotic resistance patterns documented. Here we report the first in vivo resistance evolution of a C. davisae clinical isolate in a patient with a complex hand trauma and provide insight in the resistance mechanism, leading to therapeutic implications for this pathogen. CASE PRESENTATION: Cedecea davisae was isolated from a patient with hand trauma during a first surgical debridement. Six days after primary surgical treatment and under antimicrobial treatment with amoxicillin-clavulanic acid and later cefepime, follow up cultures yielded C. davisae which demonstrated a resistance development. The susceptible parental isolate and its resistant derivative were characterized by whole genome sequencing, ampC, ompC and ompF by RT- PCR. The resistant derivative demonstrated an A224G SNP in ampD, the transcriptional regulator of ampC, leading to a His75Arg change in the corresponding AmpD protein. AmpC transcription of the resistant derivative was 362-times higher than the susceptible isolate. Transcription levels of ompF and ompC were 8.5-fold and 1.3-fold lower, respectively, in the resistant derivative. Downregulation of OmpF putatively resulted from a mutation in the presumed promoter region upstream of the dusB-Fis operon, a proposed regulator for ompF. CONCLUSIONS: This case demonstrates the in vivo resistance development of C. davisae within 7 days similar to that of the members of the Enterobacter cloacae complex. Our findings add valuable information for future therapeutic management of these opportunistic pathogens as they warrant the same empirical treatment as AmpC producers.

Molecular Epidemiology and Risk Factors for Extended-Spectrum ß-Lactamase-Producing Enterobacterales in Long-Term Care Residents.

OBJECTIVES: We aimed to assess the burden of extended-spectrum ß-lactamase (ESBL)-producing Enterobacterales in Swiss long-term care facilities (LTCFs) to describe the molecular epidemiology, describe the intrainstitutional and regional clusters of resistant pathogens, and identify independent institution- and resident-level factors associated with colonization. DESIGN: Cross-sectional study. SETTING AND PARTICIPANTS: From August to October 2019, we performed a point prevalence study among residents from 16 LTCFs in Western and Eastern Switzerland (8 per region). METHODS: Residents underwent screening for ESBL-producing Enterobacterales (ESBL-E); whole-genome sequencing (WGS) was performed. We gathered institution-level (eg, number of beds, staff-resident ratio, alcoholic hand rub consumption) and resident-level [eg, anthropometric data, time in facility, dependency, health care exposure, antibiotic treatment, proton-pump inhibitor (PPI) use] characteristics. Factors associated with colonization were identified using a generalized linear model. RESULTS: Among 1185 eligible residents, 606 (51%) consented to the study. ESBL-E prevalence was 11.6% (70/606), ranging from 1.9% to 33.3% between institutions, with a median of 12.5% in the West and 6.9% in the East (P = .03). Among 59 Escherichia coli (from 58 residents), multilocus sequence type (ST) 131 was most common (n = 43/59, 73%), predominantly its subclone H30R1 (n = 37/43, 86%). WGS data identified multiple intrainstitutional and regional clusters. Independent risk factors for ESBL carriage were previous ESBL colonization [adjusted odds ratio (aOR) 23.5, 95% confidence interval (CI) 6.6-83.8, P < .001), male gender (aOR 2.6, 95% CI 1.5-4.6, P = .002), and use of PPIs (aOR 2.2, 95% CI 1.2-3.8, P = .01). CONCLUSIONS AND IMPLICATIONS: Overall ESBL-E prevalence in Swiss LTCF residents is low. Yet, we identified several clusters of residents with identical pathogens within the same institution. This implies that particularly affected institutions might benefit from targeted infection control interventions. PPI use was the only modifiable factor associated with carriage of ESBL producers. This study adds to the growing list of adverse outcomes associated with PPIs, calling for action to restrict their use in the long-term care setting.

Antimicrobial resistance and antibiotic consumption in intensive care units, Switzerland, 2009 to 2018.

BackgroundIntensive care units (ICU) constitute a high-risk setting for antimicrobial resistance (AMR).AimWe aimed to describe secular AMR trends including meticillin-resistant Staphylococcus aureus (MRSA), glycopeptide-resistant enterococci (GRE), extended-spectrum cephalosporin-resistant Escherichia coli (ESCR-EC) and Klebsiella pneumoniae (ESCR-KP), carbapenem-resistant Enterobacterales (CRE) and Pseudomonas aeruginosa (CRPA) from Swiss ICU. We assessed time trends of antibiotic consumption and identified factors associated with CRE and CRPA.MethodsWe analysed patient isolate and antibiotic consumption data of Swiss ICU sent to the Swiss Centre for Antibiotic Resistance (2009-2018). Time trends were assessed using linear logistic regression; a mixed-effects logistic regression was used to identify factors associated with CRE and CRPA.ResultsAmong 52 ICU, MRSA decreased from 14% to 6% (p = 0.005; n = 6,465); GRE increased from 1% to 3% (p = 0.011; n = 4,776). ESCR-EC and ESCR-KP increased from 7% to 15% (p < 0.001, n = 10,648) and 5% to 11% (p = 0.002; n = 4,052), respectively. CRE, mostly Enterobacter spp., increased from 1% to 5% (p = 0.008; n = 17,987); CRPA remained stable at 27% (p = 0.759; n = 4,185). Antibiotic consumption in 58 ICU increased from 2009 to 2013 (82.5 to 97.4 defined daily doses (DDD)/100 bed-days) and declined until 2018 (78.3 DDD/100 bed-days). Total institutional antibiotic consumption was associated with detection of CRE in multivariable analysis (odds ratio per DDD: 1.01; 95% confidence interval: 1.0-1.02; p = 0.004).DiscussionIn Swiss ICU, antibiotic-resistant Enterobacterales have been steadily increasing over the last decade. The emergence of CRE, associated with institutional antibiotic consumption, is of particular concern and calls for reinforced surveillance and antibiotic stewardship in this setting.

Digital SARS-CoV-2 Detection Among Hospital Employees: Participatory Surveillance Study.

BACKGROUND: The implementation of novel techniques as a complement to traditional disease surveillance systems represents an additional opportunity for rapid analysis. OBJECTIVE: The objective of this work is to describe a web-based participatory surveillance strategy among health care workers (HCWs) in two Swiss hospitals during the first wave of COVID-19. METHODS: A prospective cohort of HCWs was recruited in March 2020 at the Cantonal Hospital of St. Gallen and the Eastern Switzerland Children's Hospital. For data analysis, we used a combination of the following techniques: locally estimated scatterplot smoothing (LOESS) regression, Spearman correlation, anomaly detection, and random forest. RESULTS: From March 23 to August 23, 2020, a total of 127,684 SMS text messages were sent, generating 90,414 valid reports among 1004 participants, achieving a weekly average of 4.5 (SD 1.9) reports per user. The symptom showing the strongest correlation with a positive polymerase chain reaction test result was loss of taste. Symptoms like red eyes or a runny nose were negatively associated with a positive test. The area under the receiver operating characteristic curve showed favorable performance of the classification tree, with an accuracy of 88% for the training data and 89% for the test data. Nevertheless, while the prediction matrix showed good specificity (80.0%), sensitivity was low (10.6%). CONCLUSIONS: Loss of taste was the symptom that was most aligned with COVID-19 activity at the population level. At the individual level-using machine learning-based random forest classification-reporting loss of taste and limb/muscle pain as well as the absence of runny nose and red eyes were the best predictors of COVID-19.

Non-occupational and occupational factors associated with specific SARS-CoV-2 antibodies among hospital workers - A multicentre cross-sectional study.

OBJECTIVES: Protecting healthcare workers (HCWs) from coronavirus disease-19 (COVID-19) is critical to preserve the functioning of healthcare systems. We therefore assessed seroprevalence and identified risk factors for severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) seropositivity in this population. METHODS: Between 22 June 22 and 15 August 2020, HCWs from institutions in northern/eastern Switzerland were screened for SARS-CoV-2 antibodies. We recorded baseline characteristics, non-occupational and occupational risk factors. We used pairwise tests of associations and multivariable logistic regression to identify factors associated with seropositivity. RESULTS: Among 4664 HCWs from 23 healthcare facilities, 139 (3%) were seropositive. Non-occupational exposures independently associated with seropositivity were contact with a COVID-19-positive household (adjusted OR 59, 95% CI 33-106), stay in a COVID-19 hotspot (aOR 2.3, 95% CI 1.2-4.2) and male sex (aOR 1.9, 95% CI 1.1-3.1). Blood group 0 vs. non-0 (aOR 0.5, 95% CI 0.3-0.8), active smoking (aOR 0.4, 95% CI 0.2-0.7), living with children <12 years (aOR 0.3, 95% CI 0.2-0.6) and being a physician (aOR 0.2, 95% CI 0.1-0.5) were associated with decreased risk. Other occupational risk factors were close contact to COVID-19 patients (aOR 2.7, 95% CI 1.4-5.4), exposure to COVID-19-positive co-workers (aOR 1.9, 95% CI 1.1-2.9), poor knowledge of standard hygiene precautions (aOR 1.9, 95% CI 1.2-2.9) and frequent visits to the hospital canteen (aOR 2.3, 95% CI 1.4-3.8). DISCUSSION: Living with COVID-19-positive households showed the strongest association with SARS-CoV-2 seropositivity. We identified several potentially modifiable work-related risk factors, which might allow mitigation of the COVID-19 risk among HCWs. The lower risk among those living with children, even after correction for multiple confounders, is remarkable and merits further study.

Prevalence of SARS-CoV-2 antibodies among Swiss hospital workers: Results of a prospective cohort study.

In this prospective cohort of 1,012 Swiss hospital employees, 3 different assays were used to screen serum for SARS-CoV-2 antibodies. Seropositivity was 1%; the positive predictive values of the lateral-flow immunoassay were 64% (IgG) and 13% (IgM). History of fever and myalgia most effectively differentiated seropositive and seronegative participants.

Successful Treatment of Acute Prostatitis Caused by Multidrug-Resistant Escherichia coli With Tigecycline Monotherapy.

We present a successful treatment, with tigecycline monotherapy, of acute prostatitis caused by multidrug-resistant Escherichia coli harboring an NDM-1 carbapemenase along with a CMY-2 cephalosporinase and a TEM ESBL.

Catheter-related bloodstream infections due to coagulase-negative staphylococci managed with catheter removal: Recurrences are rare.

Little research has been carried out on recurrences of catheter-related bloodstream infection due to coagulase-negative staphylococci (CoNS-CRBSI). The main objective of this study was to characterize patients with CoNS-CRBSI and infection recurrence after catheter removal. We included 184 CoNS-CRBSI episodes. Only 8 patients experienced recurrent bacteremia and none of them developed secondary infection of preexisting orthopedic or intravascular implant material.

Gradual in vitro Evolution of Cefepime Resistance in an ST131 Escherichia coli Strain Expressing a Plasmid-Encoded CMY-2 ß-Lactamase.

BACKGROUND: In a previous report, a clinical ST131 Escherichia coli isolate (Ec-1),producing a plasmid-encoded AmpC ß-lactamase CMY-2, evolved in vivo under cefepime (FEP) treatment to the FEP-resistant Ec-2 strain expressing an extended-spectrum ß-lactamase CMY-33. To compare factors responsible for in vitro and in vivo FEP resistance, we reproduced in vitro FEP resistance evolution in Ec-1. METHODS: FEP-resistant mutants were generated by subjecting Ec-1 (FEP MIC = 0.125 mg/L) to sub-inhibitory concentrations of FEP. MICs were obtained by broth microdilution or Etest. Strains were sequenced on an Illumina HiSeq platform. Transcriptional levels and plasmid copy numbers were determined by real-time PCR. Outer membrane proteins (OMPs) were extracted and separated by SDS-PAGE. Growth kinetics was evaluated by measuring OD450. RESULTS: The CMY-2 expressed by Ec-1 evolved to a CMY-69 (strain EC-4) by an Ala294Pro substitution after 24 passages. After 30 passages, the FEP MIC increased to 256 mg/L (strain EC-32). SDS PAGE did not reveal any lack of OMPs in the mutant strains. However, bla CMY transcription levels were up to 14-times higher than in Ec-1, which was partially explained by mutations in the upstream region of repA resulting in a higher copy number of the bla CMY-harboring IncI1 plasmid. All mutants showed a slight growth defect but no significant difference in relative growth rates compared to Ec-1. CONCLUSION: In vitro sub-inhibitory concentrations of FEP resulted in the selection of resistance mutations altering the H-10 helix of the CMY-2 and increasing the plasmid copy number. Appropriate dosing strategies may help preventing resistance evolution during treatments.

First two cases of severe multifocal infections caused by Klebsiella pneumoniae in Switzerland: characterization of an atypical non-K1/K2-serotype strain causing liver abscess and endocarditis.

BACKGROUND: We describe the first two multifocal invasive infections due to Klebsiella pneumoniae recently observed in Switzerland. METHODS: Phenotypic (MIC assays and string test) and molecular analyses (PCR/Sequencing for bla, virulence factor genes and whole genome sequencing for one strain) were performed to characterize the causative K. pneumoniae isolates. RESULTS: Both K. pneumoniae isolates (Kp1 and Kp2) were pan-susceptible to antibiotics and produced narrow-spectrum SHV ß-lactamases. However, only Kp1 was string test positive. Kp1 was of ST380 and caused liver abscess as well as pneumonia and orbital phlegmon in an Eritrean patient. It belonged to the hypervirulent capsular serotype K2 and harboured the classic virulence-associated rmpA and aerobactin genes, fulfilling both the clinical and microbiological definitions for an invasive K. pneumoniae syndrome. Kp2 was of ST1043 and caused both liver abscess and endocarditis in a Swiss patient. Moreover, it did not possess the classic virulence-associated genes. Whole genome sequencing identified less well-known virulence factors in Kp2 that might have contributed to its virulence. Among these there were genes important for intestinal colonization and/or invasion, such as genes involved in adhesion (e.g., fimABCD and mrkABCD), regulation of capsule polysaccharide biosynthesis (e.g., evgS-evgA), as well as iron uptake (iroN), energy conversion, and metabolism. DISCUSSION: This report confirms the continuous dissemination of hypervirulent K. pneumoniae strains among patients of non-Asian descent in Europe. Moreover, it highlights the genetic background of an atypical hypervirulent K. pneumoniae causing a severe invasive infection despite not possessing the classical virulence characteristics of hypermucoviscous strains.

The differential importance of mutations within AmpD in cephalosporin resistance of Enterobacter aerogenes and Enterobacter cloacae.

Mechanisms leading to carbapenem and cephalosporin resistance were sought in Enterobacter aerogenes isolates that were highly resistant to carbapenems but had no known carbapenemase. Results were compared with recent work examining carbapenem-resistant Enterobacter cloacae. Eighteen carbapenem-resistant E. aerogenes were screened for known ß-lactamase and carbapenemase genes, and novel carbapenemases were sought in whole-genome sequencing (WGS) data of the three most resistant isolates. For all isolates, ampC, ampR, ampD and the porin genes omp35 and omp36 were investigated by Sanger sequencing or from available WGS data. Expression of ampC and porin genes was measured in comparison with cephalosporin- and carbapenem-susceptible control strains by reverse transcriptase PCR, with porin translation also detected by SDS-PAGE. Loss of Omp35, primarily due to decreased transcription (up to 250×), was observed in ertapenem-resistant isolates (MICs ≥ 2 mg/L), whereas meropenem resistance (MICs ≥ 4 mg/L) was observed in those isolates also showing decreased or no production of Omp36. Loss of Omp36 was due to combinations of premature translation termination or reduced transcription. In contrast to E. cloacae, cephalosporin resistance in E. aerogenes was not associated with lesions in AmpD. High-level cefepime resistance (MIC = 32 mg/L) was caused by a novel modification in the H-10 helix of AmpC in one isolate. The differential importance of AmpD lesions in cephalosporin resistance in E. cloacae and E. aerogenes underlines the differences between these contrasting members of the Enterobacter genus. Porin loss resulted in high-level carbapenem resistance with gradual loss of Omp36, which led to high-level meropenem resistance.