Dagfinn Skaare

Multilocus sequence typing and ftsI sequencing: a powerful tool for surveillance of penicillin-binding protein 3-mediated beta-lactam resistance in nontypeable Haemophilus influenzae

BackgroundBeta-lactam resistance in Haemophilus influenzae due to ftsI mutations causing altered penicillin-binding protein 3 (PBP3) is increasing worldwide. Low-level resistant isolates with the N526K substitution (group II low-rPBP3) predominate in most geographical regions, while high-level resistant isolates with the additional S385T substitution (group III high-rPBP3) are common in Japan and South Korea.Knowledge about the molecular epidemiology of rPBP3 strains is limited. We combined multilocus sequence typing (MLST) and ftsI/PBP3 typing to study the emergence and spread of rPBP3 in nontypeable H. influenzae (NTHi) in Norway.ResultsThe prevalence of rPBP3 in a population of 795 eye, ear and respiratory isolates (99% NTHi) from 2007 was 15%. The prevalence of clinical PBP3-mediated resistance to ampicillin was 9%, compared to 2.5% three years earlier. Group II low-rPBP3 predominated (96%), with significant proportions of isolates non-susceptible to cefotaxime (6%) and meropenem (20%). Group III high-rPBP3 was identified for the first time in Northern Europe.Four MLST sequence types (ST) with characteristic, highly diverging ftsI alleles accounted for 61% of the rPBP3 isolates. The most prevalent substitution pattern (PBP3 type A) was present in 41% of rPBP3 isolates, mainly carried by ST367 and ST14. Several unrelated STs possessed identical copies of the ftsI allele encoding PBP3 type A.Infection sites, age groups, hospitalization rates and rPBP3 frequencies differed between STs and phylogenetic groups.ConclusionsThis study is the first to link ftsI alleles to STs in H. influenzae. The results indicate that horizontal gene transfer contributes to the emergence of rPBP3 by phylogeny restricted transformation.Clonally related virulent rPBP3 strains are widely disseminated and high-level resistant isolates emerge in new geographical regions, threatening current empiric antibiotic treatment. The need of continuous monitoring of beta-lactam susceptibility and a global system for molecular surveillance of rPBP3 strains is underlined. Combining MLST and ftsI/PBP3 typing is a powerful tool for this purpose.

Mutant ftsI genes in the emergence of penicillin-binding proteinmediated β-lactam resistance in Haemophilus influenzae in Norway

The most important mechanism for beta-lactam resistance in beta-lactamase-negative ampicillin-resistant (BLNAR) isolates of Haemophilus influenzae is the alteration of penicillin-binding protein 3 (PBP3) as a result of ftsI gene mutations. The present study aimed to map PBP3 alterations and to determine the correlation to beta-lactam resistance in respiratory tract isolates of H. influenzae in Norway, as well as assess the contribution of clonal spread to the emergence of PBP3-mediated resistance. Twenty-three beta-lactamase negative respiratory tract isolates with resistance to penicillins and 23 susceptible control isolates were examined by determination of beta-lactam MICs, ftsI sequencing and molecular typing by pulsed-field gel electrophoresis (PFGE). Ampicillin MIC ranges in the resistant group and the control group were 1-2 mg/L and 0.125-0.5 mg/L, respectively. All isolates in the resistant group had the PBP3 substitution Asn526-->Lys and were thus categorized as group II low-BLNAR. No control isolate met the genetic BLNAR (gBLNAR) criteria. The PBP3 substitution patterns corresponded well to those observed in previous European studies. Eighty-three percent (19/23) of the resistant isolates belonged to two clones, demonstrating the capability of low-BLNAR strains of clonal dissemination. Combined analysis of ftsI DNA sequences and PFGE patterns revealed distinctly different ftsI alleles in genetically indistinguishable isolates and identical copies of the same ftsI allele in unrelated isolates. A possible explanation of this observation is the recombinational exchange of ftsI alleles. This phenomenon, as well as the possibility of endemic European gBLNAR strains, should be further investigated.

Emergence of clonally related multidrug resistant Haemophilus influenzae with penicillin-binding protein 3-mediated resistance to extended-spectrum cephalosporins, Norway, 2006 to 2013

Resistance to cephalosporins in Haemophilus influenzae is usually caused by characteristic alterations in penicillin-binding protein 3 (PBP3), encoded by the ftsI gene. Resistance to extended-spectrum cephalosporins is associated with high-level PBP3-mediated resistance (high-rPBP3), defined by the second stage S385T substitution in addition to a first stage substitution (R517H or N526K). The third stage L389F substitution is present in some high-rPBP3 strains. High-rPBP3 H. influenzae are considered rare outside Japan and Korea. In this study, 30 high-rPBP3 isolates from Norway, collected between 2006 and 2013, were examined by serotyping, multilocus sequence typing (MLST), ftsI sequencing, detection of beta-lactamase genes and minimum inhibitory concentration (MIC) determination. MICs were interpreted according to clinical breakpoints from the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Respiratory isolates predominated (proportion: 24/30). The 30 isolates included one serotype f isolate, while the remaining 29 lacked polysaccharide capsule genes. Resistance to extended-spectrum cephalosporins (cefixime, 29 isolates/30 isolates; cefepime, 28/30; cefotaxime, 26 /30; ceftaroline, 26/30; ceftriaxone, 14/30), beta-lactamase production (11/30) and co-resistance to non-beta-lactams (trimethoprim-sulfamethoxazole, 13/30; tetracycline, 4/30; chloramphenicol, 4/30; ciprofloxacin, 3/30) was frequent. The N526K substitution in PBP3 was present in 23 of 30 isolates; these included a blood isolate which represents the first invasive S385T + N526K isolate reported from Europe. The L389F substitution, present in 16 of 30 isolates, coincided with higher beta-lactam MICs. Non-susceptibility to meropenem was frequent in S385T + L389F + N526K isolates (8/12). All 11 beta-lactamase positive isolates were TEM-1. Five clonal groups of two to 10 isolates with identical MLST-ftsI allelic profiles were observed, including the first reported high-rPBP3 clone with TEM-1 beta-lactamase and co-resistance to ciprofloxacin, tetracycline, chloramphenicol and trimethoprim-sulfamethoxazole. Prior to this study, no multidrug resistant high-rPBP3 H. influenzae had been reported in Norway. Intensified surveillance of antimicrobial resistance is needed to guide empiric therapy.

Haemophilus influenzae with Non-Beta-Lactamase-Mediated Beta-Lactam Resistance: Easy To Find but Hard To Categorize

ABSTRACT Haemophilus influenzae is a major pathogen, and beta-lactams are first-line drugs. Resistance due to altered penicillin-binding protein 3 (rPBP3) is frequent, and susceptibility testing of such strains is challenging. A collection of 154 beta-lactamase-negative isolates with a large proportion of rPBP3 (67.5%) was used to evaluate and compare Etest (Haemophilus test medium [HTM]) and disk diffusion (EUCAST method) for categorization of susceptibility to aminopenicillins and cefuroxime, using MICs generated with broth (HTM) microdilution and clinical breakpoints from CLSI and EUCAST as the gold standards. In addition, the proficiency of nine disks in screening for the rPBP3 genotype (N526K positive) was evaluated. By Etest, both essential and categorical agreement were generally poor (<70%), with high very major errors (VME) (CLSI, 13.0%; EUCAST, 34.3%) and falsely susceptible rates (FSR) (CLSI, 87.0%; EUCAST, 88.3%) for ampicillin. Ampicillin (2 μg) with adjusted (+2 mm) zone breakpoints was superior to Etest for categorization of susceptibility to ampicillin (agreement, 74.0%; VME, 11.0%; FSR, 28.3%). Conversely, Etest was superior to 30 μg cefuroxime for categorization of susceptibility to cefuroxime (agreement, 57.1% versus 60.4%; VME, 2.6% versus 9.7%; FSR, 7.1% versus 26.8%). Benzylpenicillin (1 unit) (EUCAST screening disk) and cefuroxime (5 μg) identified rPBP3 isolates with highest accuracies (95.5% and 92.2%, respectively). In conclusion, disk screening reliably detects rPBP3 H. influenzae, but false ampicillin susceptibility is frequent with routine methods. We suggest adding a comment recommending high-dose aminopenicillin therapy or the use of other agents for severe infections with screening-positive isolates that are susceptible to aminopenicillins by gradient or disk diffusion.

Prevalence of M. genitalium and U. urealyticum in urine tested for C. trachomatis.

BACKGROUND Mycoplasma genitalium and Ureaplasma urealyticum cause sexually transmitted infections. While M. genitalium is an established aetiological agent, U. urealyticum is still controversial as a pathogen. Testing for these microbes is not yet widely available in Norway, and knowledge of their prevalence is limited. In this study we have investigated the prevalence of M. genitalium and U. urealyticum in a heterogeneous population from Vestfold and Telemark. MATERIAL AND METHOD Urine samples (n = 4,665) received by the laboratory for testing for Chlamydia trachomatis in the period from February 2011 to January 2012 were subsequently tested for M. genitalium and U. urealyticum. Samples were analysed using an in-house PCR protocol. RESULTS The prevalence of C. trachomatis, M. genitalium and U. urealyticum was 11.9%, 3.6% and 17.9% respectively. M. genitalium was found most frequently in women aged 20-24 years (5.1%), while the proportion of samples positive for U. urealyticum was greatest in persons aged 15-24 years (22.8%). INTERPRETATION M. genitalium was highly prevalent in urine samples submitted for C. trachomatis testing. M. genitalium testing was requested for only a minority of the samples analysed, suggesting limited knowledge of this microbe. U. urealyticum was the most predominant microbe in the study, which may indicate that it is largely non-pathogenic.

Mycoplasma pneumoniae detection causes excess antibiotic use in Norwegian general practice: a retrospective case–control study

BACKGROUND The 2011 Mycoplasma pneumoniae epidemic in Norway resulted in many GP consultations and significantly increased the prescription of macrolide antibiotics. AIM To investigate the signs, symptoms, course, and prescription patterns of antibiotics in patients positive for M. pneumoniae compared with patients negative for M. pneumoniae. DESIGN AND SETTING A retrospective case-control study using questionnaires collected from GPs in a county in Norway. A total of 212 M. pneumoniae positive and 202 control patients were included. METHOD Descriptive statistics and logistic regression analyses were performed on the reported findings. RESULTS Forty-eight per cent of patients positive for M. pneumoniae received an antibiotic at first consultation. Another 45% in the same group received antibiotics after the polymerase chain reaction (PCR) result was known, although these patients were not clinically different from all other patients not receiving an antibiotic at first consultation. Logistic regression analysis to evaluate independent predictors for prescription of antibiotics at first consultation showed that the following factors were significantly associated: elevated C-reactive protein (CRP) level, temperature >38.0°C, pathological findings on pulmonary auscultation, and impaired general condition. Elevated CRP level, younger age, temperature >38.0°C, short duration of symptoms, and absence of rhinitis were found to be positive predictors for M. pneumoniae infection. CONCLUSION A positive PCR test for M. pneumoniae tends to trigger an antibiotic prescription, irrespective of the severity of the patients condition at first consultation. New guidelines for treatment and possibly PCR testing should be established.

Klamydiatesting i praksis – rekvirenter og pasienter

BACKGROUND Optimising the diagnostic work-up and treatment of genital chlamydia infection requires knowledge of the sampling patterns of those who order chlamydia tests. We wished to determine which groups of doctors collect specimens for chlamydia testing, and to examine the sex and age distribution of patients tested, and the proportion of positive tests, from general practitioners, gynaecologists in private practice, and youth health services. MATERIAL AND METHOD The study includes 43 465 specimens analysed for genital infection with Chlamydia trachomatis at Vestfold Hospital Trust over the period 1 January 2007 to 31 December 2011. Data from the laboratory information system were used to classify the test requisitioners. RESULTS General practitioners requisitioned 60 % of all chlamydia tests and 63 % of all positive tests. Youth health services requisitioned 13 % of all tests and 22 % of positive tests; gynaecologists in private practice, 12 % of all tests and 5 % of positive tests. Overall, 26 % of specimens were from women over the age of 30 with 2.2 % testing positive, and 82 % of these specimens were submitted by general practitioners or gynaecologists in private practice. Twenty-three per cent of specimens were from men, and 78 % of these were collected in general practice. INTERPRETATION Knowledge of who requisitions chlamydia testing and of whom is important for planning and improving chlamydia diagnosis, treatment and contact tracing. In this study from Norway, we found that doctors in general practice play a key role in diagnosing and treating chlamydia. The testing of women over the age of 30 by general practitioners and gynaecologists in private practice probably leads to unnecessary use of resources and should be reduced.