Effects of penicillin V on the faecal microbiota in patients with pharyngotonsillitis-an observational study.

Background: The intestinal microbiota functions as a reservoir of antibiotic resistance. Objectives: To evaluate penicillin V (phenoxymethylpenicillin) effects on the faecal microbiota with focus on beta-lactam resistance. Methods: We included 31 primary care patients with group A streptococcal pharyngotonsillitis treated with penicillin V for 5 (800 mg × 4) or 10 days (1000 mg × 3). Twenty-nine patients contributed with three faecal swab samples each. The faecal specimens were collected at the start of penicillin V treatment, after the last dose and at follow-up 7-9 days after completed treatment. Samples were inoculated semiquantitatively on selective screening agar plates to study beta-lactam resistance, species shifts among Enterobacterales and enterococci, and colonization with Candida spp. and Clostridioides difficile. Representative colonies were identified using MALDI-TOF. Results were analysed by non-parametric statistical methods. Results: An increase in the proportion of patients colonized with ampicillin-resistant Enterobacterales, from 52% to 86% (P = 0.007), and Enterobacterales with decreased susceptibility to third-generation cephalosporins, from 32% to 52% (P = 0.034), was observed between the first and second samples. This increase was no longer significant at follow-up. New colonization with ampicillin-resistant Enterobacterales species and non-Enterobacterales Gram-negative species was observed, and persisted at follow-up. Conclusions: Following treatment with penicillin V, we observed decreased susceptibility to ampicillin and third-generation cephalosporins, and prolonged colonization with non-Escherichia coli Gram-negative species. These findings challenge the perception that penicillin V has limited ecological effect on the intestinal microbiota, and emphasizes the importance of avoiding even narrow-spectrum antimicrobials when possible.

The clinical and microbiological efficacy of temocillin versus cefotaxime in adults with febrile urinary tract infection, and its effects on the intestinal microbiota: a randomised multicentre clinical trial in Sweden.

BACKGROUND: Use of third-generation cephalosporins, such as cefotaxime, is associated with an increased risk of selection for antimicrobial resistance, so alternative antibiotics need to be considered. The aim of the present study was to evaluate intestinal colonisation with third-generation cephalosporin-resistant pathogens following use of temocillin-an alternative antibiotic to cefotaxime that is potentially less prone to disturbing the intestinal microbiota-in empirical treatment of febrile urinary tract infection (UTI). METHODS: We did a randomised, multicentre, superiority, open-label phase 4 trial in patients who had been admitted to inpatient care in 12 Swedish hospitals with suspected or diagnosed febrile UTI (complicated or uncomplicated). To meet inclusion criteria, a patient was required to have at least one sign or symptom of pyelonephritis (ie, flank pain; costovertebral angle tenderness; and changes to urinary frequency or urgency or dysuria), a fever of 38·0°C or higher, and a positive urine dipstick (for nitrites, white blood cells, or both). Participants were also required to have an indication for intravenous antibiotic treatment. Participants were randomly assigned (1:1) to receive either 2 g temocillin or 1-2 g cefotaxime, by local investigators opening consecutive sealed randomisation envelopes that were generated centrally in advance. Both drugs were administered intravenously every 8 h. The trial was open label for investigators and patients, but those doing the microbiological analyses were masked to the groups. Participants were treated with antibiotics for 7-10 days (or up to 14 days if they had bacteraemia), at least 3 days of which were on the study drug; at day 4 and later, participants who were showing improvement could be given an oral antibiotic (ciprofloxacin, ceftibuten, cefixime, or co-trimoxazole). Patients not showing improvement were regarded as having treatment failures. Rectal swabs were collected at three timepoints: at baseline (before the first dose), after the last dose of study drug, and 7-10 days after treatment stopped. The composite primary outcome was colonisation with Enterobacterales with reduced susceptibility to third-generation cephalosporins, or colonisation with toxin-producing Clostridioides difficile, or both, to evaluate disturbance of the intestinal microbiota. The study is registered in the EU Clinical Trials Register (EudraCT 2015-003898-15). FINDINGS: Between May 20, 2016, and July 31, 2019, 207 patients were screened for eligibility, of whom 55 patients were excluded. 152 participants were randomly assigned to groups: 77 (51%) patients received temocillin, 75 (49%) patients received cefotaxime. The composite primary endpoint was met by 18 (26%) of 68 participants receiving temocillin versus 30 (48%) of 62 patients receiving cefotaxime (risk difference -22% [95% CI -42% to -3%]), showing superiority of temocillin versus cefotaxime (ie, less disturbance of the intestinal microbiota). 43 adverse events were reported in 40 (52%) of 77 patients in the temocillin group, versus 46 adverse events in 34 (45%) of 75 patients in the cefotaxime group. Most events were of mild to moderate severity. 21 (27%) patients in the temocillin and 17 (23%) patients in the cefotaxime group had an adverse event that was considered to be associated with the study drug. INTERPRETATION: Temocillin was found to be less selective than cefotaxime of Enterobacterales with reduced susceptibility to third-generation cephalosporins, and it could therefore be a favourable alternative in the empirical treatment of febrile UTI. Use of this antibiotic could reduce hospital transmission and health-care-associated infections by these pathogens. FUNDING: Public Health Agency of Sweden.

An outbreak investigation of Legionella non-pneumophila Legionnaires' disease in Sweden, April to August 2018: Gardening and use of commercial bagged soil associated with infections.

In early June 2018, an increase in non-travel-related cases of Legionella non-pneumophila Legionnaires' disease (LD) was observed in Sweden and a national outbreak investigation was started. Outbreak cases were defined as notified confirmed or probable cases of L. non-pneumophila LD, with symptom onset after 1 April 2018. From April to August 2018, 41 cases were reported, 30 of whom were identified as L. longbeachae. We conducted a case-control study with 27 cases and 182 matched controls. Results from the case-control study indicated that gardening and handling commercial bagged soil, especially dusty dry soil, were associated with disease. L. longbeachae was isolated in soils from cases' homes or gardens, but joint analysis of soil and human specimens did not identify any genetic clonality. Substantial polyclonality was noted between and within soil samples, which made finding a genetic match between soil and human specimens unlikely. Therefore, whole genome sequencing may be of limited use to confirm a specific soil as a vehicle of transmission for L. longbeachae. Handling soil for residential gardening was associated with disease and the isolation of L. longbeachae in different soils provided further evidence for Legionella non-pneumophila infection from soil.

Cross-border spread of blaNDM-1- and blaOXA-48-positive Klebsiella pneumoniae: a European collaborative analysis of whole genome sequencing and epidemiological data, 2014 to 2019.

Analysis of sequencing data for 143 blaNDM-1- and blaOXA-48-positive Klebsiella pneumoniae isolates from 13 European national collections and the public domain resulted in the identification of 15 previously undetected multi-country transmission clusters. For 10 clusters, cases had prior travel/hospitalisation history in countries outside of the European Union including Egypt, Iran, Morocco, Russia, Serbia, Tunisia and Turkey. These findings highlight the benefit of European whole genome sequencing-based surveillance and data sharing for control of antimicrobial resistance.

Antimicrobial resistance of Escherichia coli isolates from outpatient urinary tract infections in women in six European countries including Russia.

OBJECTIVES: In the Northern Dimension Antibiotic Resistance Study (NoDARS), Finland, Germany, Latvia, Poland, Russia and Sweden collected urine samples from outpatient women (aged 18-65years) with symptoms of uncomplicated urinary tract infection (UTI) to investigate the levels of antimicrobial resistance (AMR) among Escherichia coli isolates. METHODS: A total of 775 E. coli isolates from 1280 clinical urine samples were collected from October 2015 to January 2017. Antimicrobial susceptibility testing was performed and the results were interpreted according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria. RESULTS: Overall AMR rates to the commonly used antibiotics nitrofurantoin, fosfomycin and mecillinam (except for Germany that was missing a result for mecillinam) were 1.2%, 1.3% and 4.1%, respectively. The highest overall resistance rates were determined for ampicillin (39.6%), trimethoprim (23.8%), trimethoprim/sulfamethoxazole (22.4%), amoxicillin/clavulanic acid (16.7%) and ciprofloxacin (15.1%), varying significantly between countries. The rate of extended-spectrum ß-lactamase (ESBL) production was 8.7%. None of the isolates showed resistance to meropenem. CONCLUSIONS: In most cases, low AMR rates were detected against the first-line antibiotics recommended in national UTI treatment guidelines, giving support to their future use. These results also support the European Association of Urology guidelines stating that nitrofurantoin, fosfomycin and mecillinam are viable treatment options for uncomplicated UTI.

Large variation in ESBL-producing Escherichia coli carriers in six European countries including Russia.

We investigated the faecal carriage prevalence of extended-spectrum ß-lactamase production in Escherichia coli (EP-EC) and/or Klebsiella pneumoniae (EP-KP) and risk factors associated with carriage among adult study subjects in Finland, Germany, Latvia, Poland, Russia and Sweden (partner countries). The aim was to get indicative data on the prevalence of ESBL-carriage in specific populations in the region. Faecal samples were collected from four study populations and screened on ChromID-ESBL and ChromID-OXA-48 plates. Positive isolates were further characterised phenotypically. Our results show a large variation in carrier prevalence ranging from 1.6% in Latvia to 23.2% in Russia for EP-EC. For the other partner countries, the prevalence of EP-EC were in increasing numbers, 2.3% for Germany, 4.7% for Finland, 6.6% for Sweden, 8.0% for Poland and 8.1% for all partner countries in total. Carriers of EP-KP were identified only in Finland, Russia and Sweden, and the prevalence was < 2% in each of these countries. No carriers of carbapenemase-producing isolates were identified. This is the first study reporting prevalence of carriers (excluding traveller studies) for Finland, Latvia, Poland and Russia. It contributes with important information regarding the prevalence of EP-EC and EP-KP carriage in regions where studies on carriers are limited.

Emergence of vanA Enterococcus faecium in Denmark, 2005-15.

Objectives: To describe the changing epidemiology of vancomycin-resistant Enterococcus faecium and Enterococcus faecalis in clinical samples in Denmark 2005-15 according to species and van type, and, furthermore, to investigate the genetic relatedness of the clinical E. faecium isolates from 2015. Methods: During 2005-14, all clinical VRE isolates were tested for the presence of vanA/B/C genes by PCR. In 2015, all clinical VRE isolates were whole-genome sequenced. From the WGS data, the presence of van genes and MLST STs were extracted in silico . Core-genome MLST (cgMLST) analysis was performed for the vancomycin-resistant E. faecium isolates. Results: During 2005-15, 1043 vanA E. faecium , 25 vanB E. faecium , 4 vanA E. faecalis and 28 vanB E. faecalis were detected. The number of VRE was <50 isolates/year until 2012 to > 200 isolates/year in 2013-15. In 2015, 368 vanA E. faecium and 1 vanB E. faecium were detected along with 1 vanA E. faecalis and 1 vanB E. faecalis . cgMLST subdivided the 368 vanA E. faecium isolates into 33 cluster types (CTs), whereas the vanB E. faecium isolate belonged to a different CT. ST203-CT859 was most prevalent (51%), followed by ST80-CT14 (22%), ST117-CT24 (6%), ST80-CT866 (4%) and ST80-CT860 (2%). Comparison with the cgMLST.org database, previous studies and personal communications with neighbouring countries revealed that the novel cluster ST203-CT859 emerged in December 2014 and spread to the south of Sweden and the Faroe Islands during 2015. Conclusions: VRE increased in Denmark during 2005-15 due to the emergence of several vanA E. faecium clones.

Novel Aminoglycoside Resistance Transposons and Transposon-Derived Circular Forms Detected in Carbapenem-Resistant Acinetobacter baumannii Clinical Isolates.

Acinetobacter baumannii has emerged as an important opportunistic pathogen equipped with a growing number of antibiotic resistance genes. Our study investigated the molecular epidemiology and antibiotic resistance features of 28 consecutive carbapenem-resistant clinical isolates of A. baumannii collected throughout Sweden in 2012 and 2013. The isolates mainly belonged to clonal complexes (CCs) with an extensive international distribution, such as CC2 (n = 16) and CC25 (n = 7). Resistance to carbapenems was related to blaOXA-23 (20 isolates), blaOXA-24/40-like (6 isolates), blaOXA-467 (1 isolate), and ISAba1-blaOXA-69 (1 isolate). Ceftazidime resistance was associated with blaPER-7 in the CC25 isolates. Two classical point mutations were responsible for resistance to quinolones in all the isolates. Isolates with high levels of resistance to aminoglycosides carried the 16S rRNA methylase armA gene. The isolates also carried a variety of genes encoding aminoglycoside-modifying enzymes. Several novel structures involved in aminoglycoside resistance were identified, including Tn6279, ΔTn6279, Ab-ST3-aadB, and different assemblies of Tn6020 and TnaphA6. Importantly, a number of circular forms related to the IS26 or ISAba125 composite transposons were detected. The frequent occurrence of these circular forms in the populations of several isolates indicates a potential role of these circular forms in the dissemination of antibiotic resistance genes.

Carbapenemase-producing Enterobacteriaceae in Sweden 2007-2013: Experiences from seven years of systematic surveillance and mandatory reporting.

Carbapenemase-producing Enterobacteriaceae (CPE) are increasing worldwide, and are a major threat to healthcare systems. Recent European data support that many countries have interregional spread of CPE or an endemic situation. In Sweden mandatory laboratory reporting of CPE of both colonisation and infection has been practiced since 2007 and since 2012 also by treating physicians. Between 2007 and 2013, 94 cases of CPE were detected in Sweden, out of which 24 were considered to cause clinical infections (bloodstream infection (n=4), urinary tract infection (n=12), wound infection (n=4), respiratory tract infection (n=2) and catheter related (n=2). The majority were detected in the hospital setting through faecal screening or as probable colonisers in clinical cultures. Travel abroad was observed in the majority of the patients (81%), and among them 84% had been hospitalised. During the study period only two chains of transmissions in Swedish hospitals were reported, involving four patients. Klebsiella pneumoniae was the primarily isolated species (n=57) followed by Escherichia coli (n=29). blaNDM was the predominant carbapenemase gene (n=36), followed by blaOXA-48-group, blaKPC and blaVIM. In 26/94 cases (28%) isolates were categorised as possible XDR (extensively drug-resistant). CPE are increasing in Sweden, but are still at a comparably low level.

Silver resistance genes are overrepresented among Escherichia coli isolates with CTX-M production.

Members of the Enterobacteriaceae with extended-spectrum beta-lactamases (ESBLs) of the CTX-M type have disseminated rapidly in recent years and have become a threat to public health. In parallel with the CTX-M type expansion, the consumption and widespread use of silver-containing products has increased. To determine the carriage rates of silver resistance genes in different Escherichia coli populations, the presence of three silver resistance genes (silE, silP, and silS) and genes encoding CTX-M-, TEM-, and SHV-type enzymes were explored in E. coli isolates of human (n = 105) and avian (n = 111) origin. The antibiotic profiles were also determined. Isolates harboring CTX-M genes were further characterized, and phenotypic silver resistance was examined. The silE gene was present in 13 of the isolates. All of them were of human origin. Eleven of these isolates harbored ESBLs of the CTX-M type (P = 0.007), and eight of them were typed as CTX-M-15 and three as CTX-M-14. None of the silE-positive isolates was related to the O25b-ST131 clone, but 10 out of 13 belonged to the ST10 or ST58 complexes. Phenotypic silver resistance (silver nitrate MIC > 512 mg/liter) was observed after silver exposure in 12 of them, and a concomitant reduced susceptibility to piperacillin-tazobactam developed in three. In conclusion, 12% of the human E. coli isolates but none of the avian isolates harbored silver resistance genes. This indicates another route for or level of silver exposure for humans than that caused by common environmental contamination. Since silE-positive isolates were significantly more often found in CTX-M-positive isolates, it is possible that silver may exert a selective pressure on CTX-M-producing E. coli isolates.

Detection of carbapenemase-producing enterobacteriaceae in the baltic countries and st. Petersburg area.

The spread of carbapenemase-producing Enterobacteriaceae is a global problem; however, no exact data on the epidemiology of carbapenemase in the Baltic countries and St. Petersburg area is available. We aimed to evaluate the epidemiology of carbapenemase-producing Escherichia coli and Klebsiella pneumoniae in the Baltic States and St. Petersburg, Russia, and to compare the different methods for carbapenemase detection. From January to May 2012, all K. pneumoniae (n = 1983) and E. coli (n = 7774) clinical isolates from 20 institutions in Estonia, Latvia, Lithuania, and St. Petersburg, Russia were screened for carbapenem susceptibility. The IMP, VIM, GIM, NDM, KPC, and OXA-48 genes were detected using real-time PCR and the ability to hydrolyze ertapenem was determined using MALDI-TOF MS. Seventy-seven strains were found to be carbapenem nonsusceptible. From these, 15 K. pneumoniae strains hydrolyzed ertapenem and carried the bla NDM gene. All of these strains carried integron 1 and most carried integron 3 as well as genes of the CTX-M-1 group. No carbapenemase-producing E. coli or K. pneumoniae strains were found in Estonia, Latvia, or Lithuania; however, NDM-positive K. pneumoniae was present in the hospital in St. Petersburg, Russia. A MALDI-TOF MS-based assay is a suitable and cost-effective method for the initial confirmation of carbapenemase production.

Survival in the environment is a possible key factor for the expansion of Escherichia coli strains producing extended-spectrum ß-lactamases.

Acquired resistance to cephalosporins in Enterobacteriaceae is a global problem. After an outbreak at Uppsala University Hospital of extended-spectrum ß-lactamase (ESBL)-positive Klebsiella pneumoniae producing CTX-M-15, there was a shift from AmpC to ESBL production among Escherichia coli isolates. To explore the basis for this epidemiological shift, 46 E. coli isolates (ESBLs, n = 23; AmpC, n = 23) were characterized with regard to genetic relatedness, ß-lactamase, replicon and integron types, antibiotic resistance profiles, and genes encoding virulence factors. In addition, the survival in the environment and on hospital-associated materials was analysed. CTX-M-15 was the most frequent ESBL (78%). Only three (13%) of the AmpC enzymes were harboured on plasmids (CMY-2, DHA-1). Independent of plasmid-mediated beta-lactamase, IncF plasmids predominated and only class I integrons were detected. The ESBL producers carried more virulence genes (p = 0.04), exhibited a broader resistance phenotype (p = 0.01) and survived significantly longer (p = 0.03) on different materials than the AmpC-producing isolates. In conclusion, ESBL-producing isolates had properties which are likely to augment their competitiveness. Apart from antibiotic resistance and virulence factors, extended survival in the environment could be a selective trait for successful ESBL-producing E. coli strains.

A multiresistant clone of Pseudomonas aeruginosa sequence type 773 spreading in a burn unit in Orumieh, Iran.

Herein, we describe the phenotypic and genotypic characterization of a multiresistant clone of Pseudomonas aeruginosa disseminating in a burn unit in Orumieh, Iran. A total of 58 isolates of P. aeruginosa were collected during August 2007 and June 2008. Minimum inhibitory concentrations (MICs) of P. aeruginosa isolates were determined against 11 antimicrobial agents by E test. Serotyping, pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST) were used for studying the clonal relationship among the isolates. Antibiotic susceptibility testing revealed that most of the isolates were multidrug resistant and colistin was the antibiotic with the highest activity. Pseudomonas aeruginosa isolates fell into nine different serotypes, and O10 and O11 were the most common. PFGE analyses showed 12 different genotypes and 68.1% of isolates showed more than 80% similarity, indicating possible clonal relatedness. These isolates were found to belong to the same sequence type, ST773. This sequence type has earlier been reported from China, and a double locus variant of this ST has been found earlier in France in a PER-1 extended-spectrum ß-lactamase-producing P. aeruginosa.

The DiversiLab system versus pulsed-field gel electrophoresis: characterisation of extended spectrum ß-lactamase producing Escherichia coli and Klebsiella pneumoniae.

Fast and reliable epidemiological typing methods for identifying outbreaks and epidemic strains of extended spectrum ß-lactamase (ESBL) producing Enterobacteriaceae are urgently needed. The DiversiLab system (DL) has been proposed for these purposes. We compared DL to pulsed-field gel electrophoresis (PFGE) on a national collection of ESBL-producing Escherichia coli (n=258; of which 226 isolates were typeable with PFGE) and Klebsiella pneumoniae (n=48) isolated in 2007. For E. coli the Wallace coefficients showed that the probability of two isolates of the same DL type having the same PFGE type was only 19.8% and the probability of two isolates of the same PFGE type having the same DL type was 90.4%. For K. pneumoniae the Wallace coefficients showed that the probability of two isolates of the same DL type having the same PFGE type was 100% and the probability of two isolates of the same PFGE type having the same DL type was 79%, indicating that for this K. pneumoniae strain collection DL was slightly more discriminatory. Only four of 48 isolates had discordant results with the two methods. In E. coli 42% of the isolates were sequence type 131 and these isolates were related at >95% similarity with DL and at ≥60% similarity with PFGE. In summary, for E. coli DL performed well in identifying isolates related by PFGE, but overestimated the genetic relatedness in the studied collection. This indicates that DL could be a primary screening method for excluding unrelated isolates. Isolates shown to be related must be confirmed with a more discriminatory method. For K. pneumoniae, DL discriminated well but overestimated the diversity of the isolates compared to PFGE, assuming a risk of missing possible genetic relatedness.

Development of a real-time SYBRGreen PCR assay for rapid detection of acquired AmpC in Enterobacteriaceae.

INTRODUCTION: Acquired AmpC enzymes, classified as miscellaneous extended-spectrum beta-lactamase (ESBL(M)) enzymes according to a recently proposed beta-lactamase classification, are increasing according to several publications. Simple and rapid methods for detection of ESBL(M) are needed for appropriate infection control. A gel-based multiplex PCR method for acquired bla(AmpC) detection and subtype classification has been available for several years. Here, we describe a modification of the protocol to suit real-time PCR platforms and to include novel genotypes. MATERIAL AND METHODS: Clinical isolates with clavulanic acid non-reversible non-susceptibility to extended-spectrum cephalosporins were subjected to combination disk testing with cefoxitin +/- cloxacillin at Malmö University Hospital. Phenotypical AmpC production was defined as cloxacillin reversible cefoxitin resistance. In this study 51 phenotypical AmpC-producing isolates, were subjected to the acquired bla(AmpC) real-time PCR assay. The acquired blaAmpC positive isolates were further characterized by DNA sequencing of the acquired AmpC encoding gene, Pulsed-Field Gel Electrophoresis (PFGE) and PCR-based replicon typing. RESULTS AND DISCUSSION: The real-time PCR assay was able to detect and sub-classify all acquired bla(AmpC) genes described to date. The assay can be performed in less than 3h, including pre-PCR preparations. Analysis of the isolate collection resulted in 18 of 51 phenotypical AmpC-producing isolates being positive in the acquired bla(AmpC) real-time multiplex PCR assay; 17 of subtype CIT and one DHA. Sequence analysis identified 16 isolates as blaCMY-2, one as blaCMY-16 and one as blaDHA-1. Detected plasmid replicon types were I1 and B/O. Two of the E. coli isolates were identical according to PFGE and the others were unrelated.