Aspects of the antimicrobial susceptibility of Streptococcus pyogenes, Streptococcus pneumoniae and Aerococcus urinae

Streptococcus pyogenes and Streptococcus pneumoniae are important human pathogens capable of causing invasive infections which need antimicrobial treatment. Aerococcus urinae is a rare and relatively unknown causative agent in invasive infections but its clinical importance is emerging. Antimicrobial susceptibility testing (AST) is a crucial method to study the usefulness of the clinically used antimicrobial drugs and to observe trends in the prevalence of the resistance or its emergence. This thesis project aimed to analyse the antimicrobial susceptibilities of S. pyogenes and S. pneumoniae towards commonly used antimicrobial drugs and to characterize both species to gain more understanding related to their resistance. The aim was also to study bacteraemic infections caused by A. urinae and to estimate the effectiveness of the given empiric cefuroxime treatment. In addition, the objective was to determine the in vitro susceptibility of A. urinae to benzylpenicillin, cefuroxime and ceftriaxone. Species identification was performed by latex agglutination (S. pyogenes), optochin susceptibility (S. pneumoniae) and/or MALDI-TOF MS (S. pneumoniae and A. urinae). Antimicrobial susceptibilities were determined by disk diffusion, gradient diffusion (Etest) and/or broth microdilution test (Sensititre) according to the EUCAST standards. S. pyogenes isolates were characterized by emm typing and resistance gene analysis was performed by multiplex PCR to detect ermB, ermTR, and/or mefA. S. pneumoniae isolates were serotyped. Clinical bacterial isolates were found from the laboratory database using WHONET or collected particularly for the study project. Clinical data from the patients were collected retrospectively. The patient catchment area was the Helsinki metropolitan area in southern Finland representing nearly a third of the Finnish population. Susceptibility and patient data were analysed statistically by SPSS. In this thesis project we described a skin and soft tissue infection epidemic in the adult population caused by macrolide and clindamycin resistant S. pyogenes emm33 in 2012–2013. Emm33 is a rare and previously unknown emm type in Finland. Epidemic S. pyogenes emm33, a putative clone, harboured the ermTR and expressed a constitutive MLSB phenotype but was susceptible to telithromycin. S. pyogenes emm33 was also susceptible to tetracycline, doxycycline, levofloxacin, moxifloxacin, and vancomycin. Studied patients were mainly 41–60 years old and bacteraemic patients often had alcoholism and/or intravenous drug abuse. The clinicians in the HUS district were informed about the epidemic and advised in empirical treatment choices. We studied the susceptibility patterns of invasive and non-invasive S. pneumoniae isolates in 2009–2014 and showed that S. pneumoniae non-susceptibility to clinically important antimicrobial agents decreased significantly in the Helsinki metropolitan area during the four years after the 10-valent pneumococcal conjugate vaccinate (PCV10) implementation in 2010. This trend was seen especially in non-invasive isolates from <5 years old patients, where non-susceptibility decreased towards penicillin, erythromycin, clindamycin, tetracycline, and trimethoprim–sulfamethoxazole. The proportion of multidrug resistant (MDR) isolates decreased also significantly from 22% in 2009 to 6% in 2014 among these patients. S. pneumoniae serotype distributions showed that the number of the PCV10 serotypes decreased among invasive isolates. In contrast, the non-PCV10 serotypes increased among invasive isolates from ≥5 years old patients and of these, serotypes 19A (38%), 3 (13%), and 6A (13%) were the most frequent. Especially 19A is associated with high prevalence of resistance therefore making the continuous surveillance of pneumococcus essential. We retrospectively studied 141 clinical A. urinae isolates and the clinical data of 77 patients with bacteraemic A. urinae infection from 2013–2018. All A. urinae isolates were susceptible to benzylpenicillin (S ≤0.125, EUCAST breakpoints for aerococci) and cefuroxime (S ≤4, EUCAST PK/PD breakpoints) but 2–3% were resistant to ceftriaxone (R >2, EUCAST PK/PD breakpoints). Bacteraemic A. urinae patients were primarily elderly men harbouring several comorbidities. All but one patient was given antimicrobial therapy and the response was mainly good based on decreased fever and CRP. Thirty-day mortality was 17% and patients who died were on average older compared to survivors. Cefuroxime was the first empiric antimicrobial agent given to 87% of the patients and it was given equally often to patients who survived and did not survive. There were no differences between the cefuroxime MIC values of the survivors versus non-survivors. Based on these findings, A. urinae was clinically susceptible in vitro to cefuroxime and furthermore, empiric cefuroxime treatment likely covers bacteraemic infections caused by A. urinae. The results of this study made A. urinae more familiar to HUS district physicians and have improved the treatment of bacteraemic A. urinae patients.Streptococcus pyogenes eli A-ryhmän streptokokki ja Streptococcus pneumoniae eli pneumokokki ovat merkittäviä ihmisen taudinaiheuttajabakteereita. Ne voivat aiheuttaa vakavia infektioita, joiden hoitoon tarvitaan mikrobilääkkeitä. Aerococcus urinae on harvinainen ja melko tuntematon bakteeri, mutta sen merkitys taudinaiheuttajana on kasvamassa. Mikrobilääkeherkkyysmääritys on keskeinen menetelmä mikrobilääkkeiden hyödyllisyyden selvittämisessä sekä mikrobilääkkeille vastustuskykyisten eli resistenttien bakteerien esiintyvyyden ja resistenssin ilmaantuvuuden seurannassa. Tämän väitöskirjan tavoitteena oli analysoida A-ryhmän streptokokin ja pneumokokin herkkyyttä yleisesti käytetyille mikrobilääkkeille ja tutkia molempia lajeja tarkemmin resistenssiin liittyvien ilmiöiden ymmärtämiseksi. Lisäksi tavoitteena oli tutkia A. urinae -bakteerin aiheuttamia vakavia infektioita sekä niiden hoidossa käytetyn kefuroksiimi-mikrobiläkkeen käyttökelpoisuutta. Kliiniset bakteerikannat haettiin laboratorion tietokannasta tai kerättiin tätä väitöskirjaprojektia varten. Bakteerilajit tunnistettiin ja niiden mikrobilääkeherkkyydet tutkittiin yleisesti hyväksytyillä menetelmillä. Tiedot potilaista haettiin takautuvasti potilastietokannoista. Potilaat olivat kotoisin Helsingin seudulta, joka kattaa noin kolmanneksen koko Suomen väestöstä. Tässä väitöskirjassa kuvattiin makrolidi- ja klindamysiiniresistentin A-ryhmän streptokokin aiheuttama iho- ja pehmytkudosinfektioiden epidemia aikuisväestössä vuosina 2012–2013. Epidemian aiheutti harvinainen ja Suomessa aiemmin tuntematon emm33-tyyppi. HUS-alueen kliinikoita ohjeistettiin välttämään klindamysiinin käyttöä ainoana empiirisenä mikrobilääkkeenä iho- ja pehmytkudosinfektioiden hoidossa. Tutkimme pneumokokin mikrobilääkeherkkyydet vuosilta 2009–2014 ja havaitsimme, että herkkyystilanne Helsingin seudulla on merkittävästi parantunut rokotusohjelmaan vuonna 2010 käyttöönotetun 10-valenttisen pneumokokkikonjugaattirokotteen myötä. Rokotteeseen kuulumattomien pneumokokkiserotyyppien määrä kuitenkin kasvoi tutkimusajanjaksolla, minkä vuoksi pneumokokin herkkyystilannetta tärkeää seurata jatkuvasti. Tutkimme A. urinae -bakteerikantojen mikrobilääkeherkkyyksiä ja vakavaan A. urinae -infektioon sairastuneiden potilaiden kliinisiä tietoja vuosilta 2013–2018. A. urinae -kannat olivat herkkiä kefuroksiimille. Potilaat olivat suurelta osin ikääntyneitä miehiä, joilla oli useita oheissairauksia. Potilaiden vaste mikrobilääkehoidolle oli pääasiassa suotuisaa. Tutkimustulostemme perusteella kefuroksiimi soveltuu tiettyjen vakavien A. urinae -bakteerin aiheuttamien infektioiden empiiriseen hoitoon