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Genetic determinants of macrolide and tetracycline resistance in penicillin non-susceptible Streptococcus pneumoniae isolates from people living with HIV in Dar es Salaam, Tanzania

Genetic determinants of macrolide and tetracycline resistance in penicillin non-susceptible... Background Over one million yearly deaths are attributable to Streptococcus pneumoniae and people living with HIV are particularly vulnerable. Emerging penicillin non-susceptible Streptococcus pneumoniae (PNSP) challenges therapy of pneumococcal disease. The aim of this study was to determine the mechanisms of antibiotic resistance among PNSP isolates by next generation sequencing. Methods We assessed 26 PNSP isolates obtained from the nasopharynx from 537 healthy human immunodeficiency virus (HIV ) infected adults in Dar es Salaam, Tanzania, participating in the randomized clinical trial CoTrimResist (Clini- calTrials.gov identifier: NCT03087890, registered on 23rd March, 2017). Next generation whole genome sequencing on the Illumina platform was used to identify mechanisms of resistance to antibiotics among PNSP. Results Fifty percent (13/26) of PNSP were resistant to erythromycin, of these 54% (7/13) and 46% (6/13) had MLS phenotype and M phenotype respectively. All erythromycin resistant PNSP carried macrolide resistance genes; six isolates had mef(A)-msr(D), five isolates had both erm(B) and mef(A)-msr(D) while two isolates carried erm(B) alone. Isolates harboring the erm(B) gene had increased MIC (> 256 µg/mL) towards macrolides, compared to isolates without erm(B) gene (MIC 4-12 µg/mL) p < 0.001. Using the European Committee on Antimicrobial Susceptibility Testing (EUCAST ) guidelines, the prevalence of azithromycin resistance was overestimated compared to genetic cor- relates. Tetracycline resistance was detected in 13/26 (50%) of PNSP and all the 13 isolates harbored the tet(M) gene. All isolates carrying the tet(M) gene and 11/13 isolates with macrolide resistance genes were associated with the mobile genetic element Tn6009 transposon family. Of 26 PNSP isolates, serotype 3 was the most common (6/26), and sequence type ST271 accounted for 15% (4/26). Serotypes 3 and 19 displayed high-level macrolide resistance and frequently carried both macrolide and tetracycline resistance genes. Conclusion The erm(B) and mef(A)-msr(D) were common genes conferring resistance to MLS in PNSP. Resistance to tetracycline was conferred by the tet(M) gene. Resistance genes were associated with the Tn6009 transposon. *Correspondence: Joel Manyahi manyahijoel@yahoo.com Full list of author information is available at the end of the article © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Manyahi et al. Ann Clin Microbiol Antimicrob (2023) 22:16 Page 2 of 7 producing MLS phenotypes [13, 14]. Macrolide efflux Background protein A and E, efflux pumps encoded by the mef(A) and Streptococcus pneumoniae is a transient colonizer of mef(E) genes, and ribosomal mutations (23S rRNA), are the nasopharynx, with colonization peaking in the early other common causes of macrolide resistance in Strep- years of life and declining into adulthood. It is associated tococcus pneumoniae [13]. The mef(A/E) genes confer with both invasive and non-invasive pneumococcal dis- the M phenotype, exhibiting low level resistance to mac- eases. The incidence of invasive pneumococcal diseases rolides, but not resistance to lincosamides and strepto- is most prominent at the extremes of age, as well as in gramin B. The macrolide resistance genes are commonly immunocompromised hosts and people with chronic carried on mobile genetic elements, facilitating their easy respiratory tract diseases. People living with HIV are intra- and interspecies dissemination [15, 16]. The Tn916 particularly vulnerable to severe pneumococcal disease transposon family that contains the tetracycline resist- [1] Globally, Streptococcus pneumoniae is estimated to ance determinant tet(M), has frequently been reported to have caused as many as 515,000 deaths (95% uncertainty harbor macrolide resistance determinant genes [17]. interval, UI  302,000–609,000) in children aged < 5  years Macrolide resistance determinants vary with geograph- in 2015, and approximately 50% of those deaths occurred ical locations [13]. In Tanzania, where macrolides and in four countries in Africa (Nigeria, Democratic republic tetracyclines are commonly used and easily accessible of Congo) and Asia (India, Pakistan) [2]. Globally, Strep- over the counter without prescriptions, the mechanisms tococcus pneumoniae is estimated to cause 1,189,937 of resistance to these antibiotics in Streptococcus pneu- deaths (UI 690,445-1,770,660) [3]. moniae has not been studied. Therefore, we performed Before 1970, pneumococci were readily susceptible this study using whole genome sequencing to determine to nearly all relevant antibiotics, and penicillin was the mechanisms of antibiotic resistance among penicillin antibiotic of choice. In the late 1970s, pneumococci with non-susceptible Streptococcus pneumoniae isolated from non-susceptibility to penicillin emerged, resulting in Tanzania. treatment failures [4, 5]. The discovery of pneumococci resistant to penicillin shifted empirical treatment for Materials and methods suspected bacterial respiratory tract infection to mac- Bacterial isolates rolides and tetracycline. In Tanzania, standard treatment Twenty-six penicillin non-susceptible Streptococcus guidelines recommend macrolides and tetracyclines as pneumoniae were isolated by culturing nasopharyngeal first and second  line treatments, respectively, for mild swabs obtained from healthy HIV infected adults in Tan- to moderate  community acquired pneumonia caused by zania as part of the randomized clinical trial CoTrim- Streptococcus pneumoniae [6]. However, the recommen- Resist (ClinicalTrials.gov identifier: NCT03087890, dation is not based on current evidence of susceptibility registered on 23rd March, 2017). The study popula - patterns, as surveillance of the trend of antibiotic resist- tion and bacterial isolates have been described previ- ance in Streptococcus pneumoniae is limited in Tanzania. ously [18]. Streptococcus pneumoniae was identified by Data from the Network for Surveillance of Pneumococcal conventional methods including optochin disk and bile Disease in the East African Region in the pre-pneumo- susceptibility and further confirmation was done by coccal vaccination era reported a low rate of Streptococ- Matrix-Assisted Laser Desorption/Ionization-Time of cus pneumoniae resistant to erythromycin and other Flight (MALDI-TOF) mass spectrometry (MS), using the antibiotics in Tanzania [7]. Furthermore, a meta-analysis Microflex LT instrument and MALDI Biotyper 3.1 soft - of childhood pneumococcal diseases in Africa prior to ware (Bruker Daltonics, Bremen, Germany). Isolates with the widespread use of the pneumococcal capsular vac- discordant results between MALDI-TOF and conven- cine (PCV) reported a low rate of resistance to eryth- tional identification with optochin disk and bile suscepti - romycin, but a substantially higher rate of resistance to bility were omitted. tetracycline [8]. Serotyping of Streptococcus pneumoniae was per- However, post-PCV surveillance studies conducted in formed by latex agglutination (Immulex   Pneumotest well-organized settings have shown increased pneumo- Kit; SSI Diagnostica A/S, Hillerød, Denmark). coccal resistance to erythromycin and other antibiotics, partly attributed to increased consumption of macrolides Antimicrobial susceptibility testing [9–11]. The E-test strip (bioMérieux, Marcy-I-Etoile, France) was Pneumococcal resistance to macrolides is mediated by used to determine the minimum inhibitory concentra- erythromycin ribosomal methylase B (erm(B)) encoding tions (MIC) for azithromycin, erythromycin and peni- enzymes that methylate the 23S rRNA, thereby inhibit- cillin. The disk diffusion method was used to determine ing macrolide binding [12]. The erm(B) confers resist - tetracycline and clindamycin susceptibility [19]. Muller ance to macrolides, lincosamides, and Streptogramin B, M anyahi et al. Ann Clin Microbiol Antimicrob (2023) 22:16 Page 3 of 7 Hinton supplemented with 5% sheep blood agar was used For azithromycin resistance, using EUCAST break- for antimicrobial susceptibility testing, and it was incu- points (MIC > 0.5 µg/mL), all PNSP (26/26) were inter- bated at 35 °C in 5% C O for 20–24 h. The guidelines of preted as resistant, but genetic markers conferring the Clinical and Laboratory Standards Institute [19], and macrolide resistance could only be found in isolates The European Committee on Antimicrobial Susceptibil - with MIC ≥ 6  µg/mL (50%, 13/26). Using CLSI inter- ity Testing [20], were used to interpret antimicrobial sus- pretation breakpoints (MIC ≥ 2  µg/mL), 58% (15/26) ceptibility testing results. PNSP was defined according to of PNSP were interpreted as resistant to azithromycin, CLSI breakpoint interpretation [19]. while genotypic markers for macrolide resistance was found in 87% (13/15) of these isolates (Table 1). Genes conferring resistance to the group of mac- Whole genome sequencing and analysis rolides, lincosamides, and streptogramin B were Whole genome sequencing was performed using the Next observed in all the 13-erythromycin resistant PNSP iso- generation sequencing platform HiSeq X10 (Illumina, lates. The MLS phenotype (resistance to macrolides, San Diego, CA, USA) at MicrobesNG (Microbes NG, lincosamides, and streptogramin B) accounted for 54% Birmingham, UK). Quality filtering and sequencing short (7/13) while the M phenotype (resistance to macrolides, read trimming were performed by MicrobesNG using but not to lincosamides or streptogramin B) accounted SPAdes and annotated in GenBank. Short read sequences for the remaining 46% (6/13). were assembled using Unicycler at MicrobesNG. The macrolide efflux gene mef(A)-msr (D) was For allocation of multi-locus sequence typing (MLST) observed in 6 isolates. Five isolates carried both the and clonal complex, we used the online MLST database erythromycin ribosomal methylase gene erm(B) and website https:// pubml st. org/. mef(A)-msr(D), while erm(B) alone was detected in Identification of acquired resistance was performed only two isolates. Isolates carrying  the erm(B) gene using the web-based platform ResFinder v3.2 of the had increased MIC for both erythromycin and azithro- Center for Genomic Epidemiology (http:// www. genom mycin, > 256  µg/mL, compared to isolates lacking  the icepi demio logy. org/). erm(B) gene (MIC 4-12 µg/mL), p < 0.001. For identification of mobile genetic elements and their The mef(A)-msr(D) gene predicted phenotypic resist- related acquired antimicrobial resistance we used the ance to macrolides, but at low MIC values (4–12 µg/mL Center for Genomic Epidemiology MobileElement Finder for erythromycin) and (6–96  µg/mL for azithromycin). v1.0.3 (http:// www. genom icepi demio logy. org/). Carrying the mef(A)-msr(D) gene alone did not predict This Whole Genome Shotgun project has been depos - phenotypic resistance to lincosamides (clindamycin) ited at DDBJ/ENA/GenBank under the BioProject num- and the six isolates carrying mef(A)-msr(D) were all ber PRJNA918594. phenotypically susceptible to clindamycin. Disc diffusion results showed that 13/26 (50%) of Statistical analysis PNSP isolates were resistant to tetracyclines. All the Categorical variables were presented in frequencies, per- 13-tetracycline resistant PNSP isolates carried the centages, and proportions. Categorical variables were tet(M) gene which confers resistance to tetracyclines. compared using chi square test. A p-value < 0.05 was con- Three PNSP isolates harbored cat (pC194) which con - sidered as threshold for statistical significance. Statistical fers resistance to chloramphenicol. analysis was performed using STATA version 16 (College The Tn6009-like element was detected in 13 PNSP. Station, TX). All tetracycline resistant PNSP were associated with a Tn6009 like element, while 12/13 of the erythromycin Results resistant PNSP had a Tn6009 like element. Twelve out A total of 26 penicillin non-susceptible Streptococcus of 13 tetracycline-resistant PNSP isolates were associ- pneumoniae were analyzed by whole genome sequenc- ated with plasmid replicon type repUS43. ing. Resistance to both macrolides and tetracyclines was Serotype 3 was the  most common, followed by sero- observed in 12/26 (46%) of PNSP isolates. type19  and 35B.  The majority of serotype  3 and 19 Phenotypic results using both EUCAST and CLSI PNSP displayed high level macrolide resistance and breakpoint interpretation showed that 13/26 (50%) of carried erm(B) and tet(M) genes. MLST analysis iden- PNSP isolates were resistant to macrolides (erythro- tified seventeen different sequence types (ST), among mycin). Phenotypic resistance to erythromycin was in which  ST271 accounted for 15% (4/26), followed by concordance with genotypic resistance determinants as ST172 (12%, 3/26) and ST14821 (8%, 2/26). The ST271 shown in Table 1. isolate  belonged to serotype 3 and carried multiple resistance-determinant genes. Manyahi et al. Ann Clin Microbiol Antimicrob (2023) 22:16 Page 4 of 7 Table 1 Phenotypic and Genotypic characteristics of penicillin non-susceptible Streptococcus pneumoniae Strain number Isolation Year Serotype ST PEN AZT ERY MLS genes TET Disc TET Plasmid Tn CHL MIC MIC MIC diffusion genes 2002c 2019 19F 5339 0.25 1.5 0.19 – – S – – – – 2145c 2019 34 5258 0.032 2 0.19 – – – S – – – 258d 2019 35B 6b3a 0.125 96 8 – mef(A) Msr(D) R Tet(M) repUS43 6009 – 469c 2018 35B 172 0.38 32 12 – mef(A) Msr(D) S – – – – 2010a 2018 19A 847 0.50 256 256 erm(B) – R Tet(M) repUS43 6009 - 263c 2018 6A 3460 0.125 1 0.125 – – – S – – – – 57c 2017 3 271 1.5 256 256 erm(B) mef(A) Msr(D) R Tet(M) repUS43 6009 – 2267a 2018 3 271 0.25 256 256 erm(B) mef(A) Msr(D) R Tet(M) repUS43 6009 – 90d 2018 15A 0c86 0.25 1 0.094 – – – S – – – 498d 2018 11A 14821 0.25 6 4 mef(A) Msr(D) R Tet(M) repUS43 6009 Cat(pC194) 116d 2018 11A 14821 0.19 32 4 mef(A) msr(D) R tet(M) repUS43 6009 Cat(pC194) 380a 2017 38 6103 0.25 1 0.094 – – – S – – – – 2071a 2018 3 2054 0.25 0.5 0.064 – – – S – rep36 – – 2019b 2018 4 9a19 0.50 1 0.125 – – – S – – – – 2267b 2018 3 271 0.75 256 256 erm(B) mef(A) msr(D) R tet(M) repUS43 6009 – 2052d 2019 3 700 0.38 0.75 0.094 – – – R tet(M) repUS43 6009 219a 2017 35B 172 0.25 1 0.094 – – – S – – – – 268a 2017 23B 6fe5 0.25 0.75 0.125 – – – S – – – – 2034b 2018 3 271 0.38 256 256 erm(B) mef(A) msr(D) R tet(M) repUS43 6009 393a 2017 35B 172 0.25 1 0.094 – – – S – – – – 2164a 2018 19F cf17 0.25 32 6 – mef(A) msr(D) R tet(M) rep13 6009 Cat(pC194) 272b 2017 19A 0c11 0.25 256 256 erm(B) – – R tet(M) repUS43 6009 – 258b 2017 21 cdc8 0.047 1 0.125 – – – S – – – – 252d 2017 46 15772 0.016 24 8 – mef(A) msr(D) R tet(M) repUS43 6009 – 2014d 2019 19F 8678 0.75 256 256 erm(B) mef(A) msr(D) R tet(M) repUS43 6009 – 369b 2018 23F 1188 0.25 2 0.125 – – – S – – – erm(B) Macrolide, Lincosamide and Streptogramin B resistance, mef(A) Macrolide resistance, msr(D) Macrolide resistance, tet(M) Tetracycline resistance, cat(pC194) Chloramphenicol resistance, AZT Azithromycin, ERY Erythromycin, PEN Penicillin, TET Tetracycline, CHL Chloramphenicol, ST sequence typing, MIC minimum inhibitory concentration M anyahi et al. Ann Clin Microbiol Antimicrob (2023) 22:16 Page 5 of 7 Discussion The erm(B) gene has been reported as the most com- We observed a discrepancy in azithromycin susceptibility mon macrolide resistance determinant in Streptococcus depending on whether using breakpoints from EUCAST pneumoniae in studies from Africa [14, 21, 22] and some or CLSI guidelines for interpretation. All 13 PNSP iso- part of Asia [11]. Macrolide resistance genes in Strepto- lates harboring genetic determinants for macrolide coccus pneumoniae have marked geographical variability resistance were correctly identified as resistant to eryth - [13]. The mef(A) has been reported to be the predomi - romycin and azithromycin (all with MIC ≥ 4  µg/mL) nant mechanism of pneumococcal macrolide resistance regardless of which breakpoints were used (sensitivity in North America and some parts of Europe [13]. Our 100%, 13/13). Using the EUCAST breakpoints appeared study found the macrolide efflux genes mef(A)/msr(D) to to overestimate azithromycin resistance, as all 13 eryth- be more prevalent (11/13) than the erm(B) genes (7/13). romycin-susceptible PNSP without genetic determinants Still, considering the high number of isolates harbor- of macrolide resistance were interpreted as resistant to ing both types of resistance genes (5/13), the dominant azithromycin (MIC-values from 0.5 to 2, specificity 0%, MLS phenotype was more frequent (7/13, erm(B) with 0/13). Using CLSI breakpoints only misclassified two or without mef(A)/msr(D)) than the M-phenotype (6/13, such isolates (MIC 2  µg/mL, specificity 85%, 11/13). mef(A)/msr(D) alone). Therefore, relying on current EUCAST guidelines Previous studies have shown that tetracycline and mac- appears to overestimate azithromycin resistance and rolide resistance genes are carried on mobile genetic ele- could in the clinical perspective lead to unnecessary use ments, composite conjugative transposons, Tn916-like of more broad-spectrum antibiotics. Our findings sug - elements, which facilitate their dissemination between gest that the EUCAST guidelines currently use a too different bacteria [23, 24]. Tn916 and Tn917-like com- low cutoff for MIC-values for azithromycin resistance in posite elements have been documented to facilitate dis- pneumococci. semination of erm(B) and mef(A/E) in Streptococcus PNSP susceptibility to erythromycin, on the other pneumoniae [17]. However, our study found a Tn6009- hand, was similar using both EUCAST and CLSI break- like element in all 13 PNSP isolates carrying the tet(M) points, and the phenotypic findings correlated well with gene which confers resistance to tetracycline, and in the identified genotypic resistance markers. To avoid var - 12/13 (92%) of PNSP isolates carrying macrolide resist- iations in interpretation, our findings call for AST guide - ance determinants. Tn6009 is a member of the Tn916– lines to be harmonized. Both CLSI and EUCAST state Tn1545  transposon family previously detected in that erythromycin susceptibility can predict susceptibil- Gram-positive and Gram-negative bacteria [25]. Tn6009 ity  to clarithromycin, azithromycin, dirithromycin, and has been reported to carry genes conferring resistance roxithromycin [19, 20]. Because almost all PNSP resistant against tetracycline tet(M), and inorganic and organic to erythromycin carried genetic determinants for mac- mercury [25]. Through horizontal gene transfer, the con - rolide resistance, our study supports that erythromycin jugative mobile elements enable bacteria to acquire and determines susceptibility to other macrolides. disseminate DNA between related and unrelated bacte- In Tanzania, macrolides are commonly used to treat ria. The presence of transposons containing macrolide respiratory tract infections. In the treatment of commu- and tetracycline resistance genes in PNSP in our study nity-acquired pneumonia, erythromycin and azithro- could indicate an increased risk of dissemination of these mycin are used as first and second line treatment, resistance determinants. respectively [6]. In this study, however, we observed that 50% and 58% of PNSP were resistant to erythromycin and azithromycin, respectively. Consequently, macrolides Conclusion appear potentially ineffective for treating PNSP infec - Macrolides and tetracyclines have only about 50% chance tions in this setting. This calls for prudent use of antibi - of being effective against PNSPs recovered from naso - otics including the use of narrow-spectrum penicillin. pharynx from people living with HIV in Dar es Salaam. But for treatment failure or infections likely caused by The erm(B) and mef(A)-msr(D) were common genes con- resistant pneumococci/PNSP,  options are difficult, with ferring resistance to macrolides and clindamycin, while azithromycin, the currently preferred treatment covering resistance to tetracycline was conferred by  the tet(M) just half of the PNSP. gene. Detection of the  composite conjugate transpo- The most common phenotype was MLS and isolates son Tn6009 associated with macrolides and tetracycline with this phenotype harbored the erm(B) gene confer- genes could indicate the possibility of horizontal transfer ring a high level of resistance to macrolides (> 256  µg/ of resistant genes. Using EUCAST guidelines for inter- mL) and clindamycin. All but two isolates with the ML S pretation overestimates azithromycin resistance in PNSP phenotype carried the mef(A) and msr(D) genes as well. compared to genetic correlates of resistance. Manyahi et al. Ann Clin Microbiol Antimicrob (2023) 22:16 Page 6 of 7 Acknowledgements a systematic analysis for the Global Burden of Disease Study 2016. Authors thank members of the bacteriology research laboratory of the Lancet Infect Dis. 2018;18(11):1191–210. https:// doi. org/ 10. 1016/ Muhimbili University of Health and Allied Science in Dar es Salaam, Tanzania, s1473- 3099(18) 30310-4. for their assistance during data collection and preliminary laboratory 4. Klugman KP. Pneumococcal resistance to antibiotics. Clin Microbiol procedures. Rev. 1990;3(2):171–96. https:// doi. org/ 10. 1128/ cmr.3. 2. 171. 5. Tleyjeh IM, Tlaygeh HM, Hejal R, Montori VM, Baddour LM. The impact Author contributions of penicillin resistance on short-term mortality in hospitalized adults BB and NL conceived the study. JM collected study data. JM and SM per- with pneumococcal pneumonia: a systematic review and meta-analy- formed the microbiological investigations. BB and JM performed statistical sis. Clin Infect Dis. 2006;42(6):788–97. https:// doi. org/ 10. 1086/ 500140. analysis. JM drafted the manuscript. BB, SJM, and NL revised the manuscript. 6. Tanzania standard treatment guidelines & national essential medicines All authors approved the final version. list. STG/NEMLIT 2021. Ministry of Health, Dar es Salaam, Tanzania 7. Mudhune S, Wamae M. Report on invasive disease and meningitis due to Funding Haemophilus influenzae and Streptococcus pneumonia from the Network This research was supported by (i) Helse Bergen HF, Haukeland University for Surveillance of Pneumococcal Disease in the East African Region. Clin Hospital, Bergen, Norway, project number 912132, , (ii) National Advisory Unit Infect Dis. 2009;48(Suppl 2):S147–52. https:// doi. org/ 10. 1086/ 596494. on Tropical Infectious Diseases, Haukeland University Hospital, Bergen, Nor- 8. Iroh Tam PY, Thielen BK, Obaro SK, Brearley AM, Kaizer AM, Chu H, et al. way, (iii) CAMRIA - Combatting Anti-Microbial Resistance with Interdisciplinary Childhood pneumococcal disease in Africa—a systematic review and Approaches, Centre for Antimicrobial Resistance in Western Norway, funded meta-analysis of incidence, serotype distribution, and antimicrobial sus- by Trond Mohn Stiftelse, grant number TMS2020TMT11, and (iv) STRESST - ceptibility. Vaccine. 2017;35(15):1817–27. https:// doi. org/ 10. 1016/j. vacci Antimicrobial Stewardship in Hospitals, Resistance Selection and Transfer in a ne. 2017. 02. 045. One Health Context, Liverpool School of Tropical Medicine, Liverpool, United 9. Sader HS, Mendes RE, Le J, Denys G, Flamm RK, Jones RN. Antimicrobial Kingdom, funded by JPIAMR grant number NFR333432. Susceptibility of Streptococcus pneumoniae from North America, Europe, Latin America, and the Asia-Pacific Region: results from 20 years of the Availability of data and materials SENTRY antimicrobial surveillance program (1997–2016). Open Forum Data are available on reasonable request. Infect Dis. 2019;6(Suppl 1):S14-s23. https:// doi. org/ 10. 1093/ ofid/ ofy263. 10. Yu YY, Xie XH, Ren L, Deng Y, Gao Y, Zhang Y, et al. Epidemiological char- acteristics of nasopharyngeal Streptococcus pneumoniae strains among Declarations children with pneumonia in Chongqing, China. Sci Rep. 2019;9(1):3324. https:// doi. org/ 10. 1038/ s41598- 019- 40088-6. Ethics approval and consent to participate 11. Beheshti M, Jabalameli F, Feizabadi MM, Hahsemi FB, Beigverdi R, Ethical approval to conduct the study were obtained from Muhimbili Univer- Emaneini M. Molecular characterization, antibiotic resistance pattern and sity of Heath and Allied Sciences Senate Research and Publications Committee capsular types of invasive Streptococcus pneumoniae isolated from clinical (Ref. No. 2015-10-27/AEC/Vol.X/54), National Health Research Ethics Commit- samples in Tehran, Iran. BMC Microbiol. 2020;20(1):167. https:// doi. org/ 10. tee (Ref. No. NIMRlHQ/R. SaJVol. 1X12144), Tanzania Medicines and Medical 1186/ s12866- 020- 01855-y. Devices Authority (Ref. No. TZ16CT007), and Regional Committee for Medical 12. Schroeder MR, Lohsen S, Chancey ST, Stephens DS. High-Level macrolide and Health Research Ethics of Western Norway (Ref. No. REK2015/540). resistance due to the mega element [mef(E)/mel] in Streptococcus pneu- Written informed consent was obtained from each study participant before moniae. Front Microbiol. 2019;10:868. https:// doi. org/ 10. 3389/ fmicb. 2019. the enrollment in the study. 13. Schroeder MR, Stephens DS. Macrolide resistance in Streptococcus pneu- Consent for publication moniae. Front Cell Infect Microbiol. 2016;6:98. https:// doi. org/ 10. 3389/ Consent to publish was obtained from National Health Research Ethics Com- fcimb. 2016. 00098. mittee (Ref. No. NIMRlHQ/R. SaJVol. 1X12144). 14. Raddaoui A, Tanfous FB, Chebbi Y, Achour W, Baaboura R, Benhassen A. High prevalence of multidrug-resistant international clones among Competing interests macrolide-resistant Streptococcus pneumoniae isolates in immunocom- Authors declare that they have no competing interests. promised patients in Tunisia. Int J Antimicrob Agents. 2018;52(6):893–7. https:// doi. org/ 10. 1016/j. ijant imicag. 2018. 04. 015. Author details 15. Korona-Glowniak I, Siwiec R, Malm A. Resistance determinants and their Department of Clinical Science, University of Bergen, Bergen, Norway. association with different transposons in the antibiotic-resistant Strepto - National Advisory Unit for Tropical Infectious Diseases, Department of Medi- coccus pneumoniae. Biomed Res Int. 2015;2015:836496. https:// doi. org/ cine, Haukeland University Hospital, Bergen, Norway. Department of Micro- 10. 1155/ 2015/ 836496. biology and Immunology, Muhimbili University of Health and Allied Sciences, 16. Talebi M, Azadegan A, Sadeghi J, Ahmadi A, Ghanei M, Katouli M, et al. P.O. Box 65001, Dar es Salaam, Tanzania. Determination of characteristics of erythromycin resistant Strep- tococcus pneumoniae with preferred PCV usage in Iran. PLoS ONE. Received: 5 September 2022 Accepted: 9 February 2023 2016;11(12):e0167803. https:// doi. org/ 10. 1371/ journ al. pone. 01678 03. 17. Chancey ST, Agrawal S, Schroeder MR, Farley MM, Tettelin H, Stephens DS. Composite mobile genetic elements disseminating macrolide resistance in Streptococcus pneumoniae. Front Microbiol. 2015;6:26. https:// doi. org/ 10. 3389/ fmicb. 2015. 00026. References 18. Manyahi J, Moyo S, Aboud S, Langeland N, Blomberg B. High rate of 1. Madhi SA, Petersen K, Madhi A, Khoosal M, Klugman KP. Increased antimicrobial resistance and multiple mutations in the dihydrofolate disease burden and antibiotic resistance of bacteria causing severe reductase gene among Streptococcus pneumoniae isolated from HIV- community-acquired lower respiratory tract infections in human infected adults in a community setting in Tanzania. J Glob Antimicrob immunodeficiency virus type 1-infected children. Clin Infect Dis. Resist. 2020;22:749–53. https:// doi. org/ 10. 1016/j. jgar. 2020. 06. 026. 2000;31(1):170–6. https:// doi. org/ 10. 1086/ 313925. 19. Clinical and Laboratory Standards Institute. Performance standards for 2. Wahl B, O’Brien KL, Greenbaum A, Majumder A, Liu L, Chu Y, et al. antimicrobial susceptibility testing; twenty-eighth informational sup- Burden of Streptococcus pneumoniae and Haemophilus influenzae plement CLSI document M100–S20. Wayne, PA: Clinical and Laboratory type b disease in children in the era of conjugate vaccines: global, Standards Institute; 2018. regional, and national estimates for 2000–15. Lancet Glob Health. 20. The European Committee on Antimicrobial Susceptibility Testing. Break- 2018;6(7):e744–57. https:// doi. org/ 10. 1016/ s2214- 109x(18) 30247-x. point tables for interpretation of MICs and zone diameters. Version 11.0, 3. Estimates of the global, regional, and national morbidity, mortality, and 2021. http:// www. eucast. org. aetiologies of lower respiratory infections in 195 countries, 1990–2016: M anyahi et al. Ann Clin Microbiol Antimicrob (2023) 22:16 Page 7 of 7 21. Midouni Ayadi B, Mehiri E, Draoui H, Ghariani A, Essalah L, Raoult D, et al. Phenotypic and molecular characterization of macrolide resistance mechanisms among Streptococcus pneumoniae isolated in Tunisia. J Med Microbiol. 2020;69(4):505–20. https:// doi. org/ 10. 1099/ jmm.0. 001151. 22. Diawara I, Zerouali K, Katfy K, Barguigua A, Belabbes H, Timinouni M, et al. Phenotypic and genotypic characterization of Streptococcus pneumo- niae resistant to macrolide in Casablanca, Morocco. Infect Genet Evol. 2016;40:200–4. https:// doi. org/ 10. 1016/j. meegid. 2016. 03. 003. 23. Roberts AP, Mullany P. Tn916-like genetic elements: a diverse group of modular mobile elements conferring antibiotic resistance. FEMS Micro- biol Rev. 2011;35(5):856–71. https:// doi. org/ 10. 1111/j. 1574- 6976. 2011. 00283.x. 24. Sansevere EA, Robinson DA. Staphylococci on ICE: overlooked agents of horizontal gene transfer. Mob Genet Elements. 2017;7(4):1–10. https:// doi. org/ 10. 1080/ 21592 56x. 2017. 13684 33. 25. Soge OO, Beck NK, White TM, No DB, Roberts MC. A novel transposon, Tn6009, composed of a Tn916 element linked with a Staphylococcus aureus mer operon. J Antimicrob Chemother. 2008;62(4):674–80. https:// doi. org/ 10. 1093/ jac/ dkn255. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Re Read ady y to to submit y submit your our re researc search h ? Choose BMC and benefit fr ? Choose BMC and benefit from om: : fast, convenient online submission thorough peer review by experienced researchers in your field rapid publication on acceptance support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year At BMC, research is always in progress. 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Genetic determinants of macrolide and tetracycline resistance in penicillin non-susceptible Streptococcus pneumoniae isolates from people living with HIV in Dar es Salaam, Tanzania

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Abstract

Background Over one million yearly deaths are attributable to Streptococcus pneumoniae and people living with HIV are particularly vulnerable. Emerging penicillin non-susceptible Streptococcus pneumoniae (PNSP) challenges therapy of pneumococcal disease. The aim of this study was to determine the mechanisms of antibiotic resistance among PNSP isolates by next generation sequencing. Methods We assessed 26 PNSP isolates obtained from the nasopharynx from 537 healthy human immunodeficiency virus (HIV ) infected adults in Dar es Salaam, Tanzania, participating in the randomized clinical trial CoTrimResist (Clini- calTrials.gov identifier: NCT03087890, registered on 23rd March, 2017). Next generation whole genome sequencing on the Illumina platform was used to identify mechanisms of resistance to antibiotics among PNSP. Results Fifty percent (13/26) of PNSP were resistant to erythromycin, of these 54% (7/13) and 46% (6/13) had MLS phenotype and M phenotype respectively. All erythromycin resistant PNSP carried macrolide resistance genes; six isolates had mef(A)-msr(D), five isolates had both erm(B) and mef(A)-msr(D) while two isolates carried erm(B) alone. Isolates harboring the erm(B) gene had increased MIC (> 256 µg/mL) towards macrolides, compared to isolates without erm(B) gene (MIC 4-12 µg/mL) p < 0.001. Using the European Committee on Antimicrobial Susceptibility Testing (EUCAST ) guidelines, the prevalence of azithromycin resistance was overestimated compared to genetic cor- relates. Tetracycline resistance was detected in 13/26 (50%) of PNSP and all the 13 isolates harbored the tet(M) gene. All isolates carrying the tet(M) gene and 11/13 isolates with macrolide resistance genes were associated with the mobile genetic element Tn6009 transposon family. Of 26 PNSP isolates, serotype 3 was the most common (6/26), and sequence type ST271 accounted for 15% (4/26). Serotypes 3 and 19 displayed high-level macrolide resistance and frequently carried both macrolide and tetracycline resistance genes. Conclusion The erm(B) and mef(A)-msr(D) were common genes conferring resistance to MLS in PNSP. Resistance to tetracycline was conferred by the tet(M) gene. Resistance genes were associated with the Tn6009 transposon. *Correspondence: Joel Manyahi manyahijoel@yahoo.com Full list of author information is available at the end of the article © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Manyahi et al. Ann Clin Microbiol Antimicrob (2023) 22:16 Page 2 of 7 producing MLS phenotypes [13, 14]. Macrolide efflux Background protein A and E, efflux pumps encoded by the mef(A) and Streptococcus pneumoniae is a transient colonizer of mef(E) genes, and ribosomal mutations (23S rRNA), are the nasopharynx, with colonization peaking in the early other common causes of macrolide resistance in Strep- years of life and declining into adulthood. It is associated tococcus pneumoniae [13]. The mef(A/E) genes confer with both invasive and non-invasive pneumococcal dis- the M phenotype, exhibiting low level resistance to mac- eases. The incidence of invasive pneumococcal diseases rolides, but not resistance to lincosamides and strepto- is most prominent at the extremes of age, as well as in gramin B. The macrolide resistance genes are commonly immunocompromised hosts and people with chronic carried on mobile genetic elements, facilitating their easy respiratory tract diseases. People living with HIV are intra- and interspecies dissemination [15, 16]. The Tn916 particularly vulnerable to severe pneumococcal disease transposon family that contains the tetracycline resist- [1] Globally, Streptococcus pneumoniae is estimated to ance determinant tet(M), has frequently been reported to have caused as many as 515,000 deaths (95% uncertainty harbor macrolide resistance determinant genes [17]. interval, UI  302,000–609,000) in children aged < 5  years Macrolide resistance determinants vary with geograph- in 2015, and approximately 50% of those deaths occurred ical locations [13]. In Tanzania, where macrolides and in four countries in Africa (Nigeria, Democratic republic tetracyclines are commonly used and easily accessible of Congo) and Asia (India, Pakistan) [2]. Globally, Strep- over the counter without prescriptions, the mechanisms tococcus pneumoniae is estimated to cause 1,189,937 of resistance to these antibiotics in Streptococcus pneu- deaths (UI 690,445-1,770,660) [3]. moniae has not been studied. Therefore, we performed Before 1970, pneumococci were readily susceptible this study using whole genome sequencing to determine to nearly all relevant antibiotics, and penicillin was the mechanisms of antibiotic resistance among penicillin antibiotic of choice. In the late 1970s, pneumococci with non-susceptible Streptococcus pneumoniae isolated from non-susceptibility to penicillin emerged, resulting in Tanzania. treatment failures [4, 5]. The discovery of pneumococci resistant to penicillin shifted empirical treatment for Materials and methods suspected bacterial respiratory tract infection to mac- Bacterial isolates rolides and tetracycline. In Tanzania, standard treatment Twenty-six penicillin non-susceptible Streptococcus guidelines recommend macrolides and tetracyclines as pneumoniae were isolated by culturing nasopharyngeal first and second  line treatments, respectively, for mild swabs obtained from healthy HIV infected adults in Tan- to moderate  community acquired pneumonia caused by zania as part of the randomized clinical trial CoTrim- Streptococcus pneumoniae [6]. However, the recommen- Resist (ClinicalTrials.gov identifier: NCT03087890, dation is not based on current evidence of susceptibility registered on 23rd March, 2017). The study popula - patterns, as surveillance of the trend of antibiotic resist- tion and bacterial isolates have been described previ- ance in Streptococcus pneumoniae is limited in Tanzania. ously [18]. Streptococcus pneumoniae was identified by Data from the Network for Surveillance of Pneumococcal conventional methods including optochin disk and bile Disease in the East African Region in the pre-pneumo- susceptibility and further confirmation was done by coccal vaccination era reported a low rate of Streptococ- Matrix-Assisted Laser Desorption/Ionization-Time of cus pneumoniae resistant to erythromycin and other Flight (MALDI-TOF) mass spectrometry (MS), using the antibiotics in Tanzania [7]. Furthermore, a meta-analysis Microflex LT instrument and MALDI Biotyper 3.1 soft - of childhood pneumococcal diseases in Africa prior to ware (Bruker Daltonics, Bremen, Germany). Isolates with the widespread use of the pneumococcal capsular vac- discordant results between MALDI-TOF and conven- cine (PCV) reported a low rate of resistance to eryth- tional identification with optochin disk and bile suscepti - romycin, but a substantially higher rate of resistance to bility were omitted. tetracycline [8]. Serotyping of Streptococcus pneumoniae was per- However, post-PCV surveillance studies conducted in formed by latex agglutination (Immulex   Pneumotest well-organized settings have shown increased pneumo- Kit; SSI Diagnostica A/S, Hillerød, Denmark). coccal resistance to erythromycin and other antibiotics, partly attributed to increased consumption of macrolides Antimicrobial susceptibility testing [9–11]. The E-test strip (bioMérieux, Marcy-I-Etoile, France) was Pneumococcal resistance to macrolides is mediated by used to determine the minimum inhibitory concentra- erythromycin ribosomal methylase B (erm(B)) encoding tions (MIC) for azithromycin, erythromycin and peni- enzymes that methylate the 23S rRNA, thereby inhibit- cillin. The disk diffusion method was used to determine ing macrolide binding [12]. The erm(B) confers resist - tetracycline and clindamycin susceptibility [19]. Muller ance to macrolides, lincosamides, and Streptogramin B, M anyahi et al. Ann Clin Microbiol Antimicrob (2023) 22:16 Page 3 of 7 Hinton supplemented with 5% sheep blood agar was used For azithromycin resistance, using EUCAST break- for antimicrobial susceptibility testing, and it was incu- points (MIC > 0.5 µg/mL), all PNSP (26/26) were inter- bated at 35 °C in 5% C O for 20–24 h. The guidelines of preted as resistant, but genetic markers conferring the Clinical and Laboratory Standards Institute [19], and macrolide resistance could only be found in isolates The European Committee on Antimicrobial Susceptibil - with MIC ≥ 6  µg/mL (50%, 13/26). Using CLSI inter- ity Testing [20], were used to interpret antimicrobial sus- pretation breakpoints (MIC ≥ 2  µg/mL), 58% (15/26) ceptibility testing results. PNSP was defined according to of PNSP were interpreted as resistant to azithromycin, CLSI breakpoint interpretation [19]. while genotypic markers for macrolide resistance was found in 87% (13/15) of these isolates (Table 1). Genes conferring resistance to the group of mac- Whole genome sequencing and analysis rolides, lincosamides, and streptogramin B were Whole genome sequencing was performed using the Next observed in all the 13-erythromycin resistant PNSP iso- generation sequencing platform HiSeq X10 (Illumina, lates. The MLS phenotype (resistance to macrolides, San Diego, CA, USA) at MicrobesNG (Microbes NG, lincosamides, and streptogramin B) accounted for 54% Birmingham, UK). Quality filtering and sequencing short (7/13) while the M phenotype (resistance to macrolides, read trimming were performed by MicrobesNG using but not to lincosamides or streptogramin B) accounted SPAdes and annotated in GenBank. Short read sequences for the remaining 46% (6/13). were assembled using Unicycler at MicrobesNG. The macrolide efflux gene mef(A)-msr (D) was For allocation of multi-locus sequence typing (MLST) observed in 6 isolates. Five isolates carried both the and clonal complex, we used the online MLST database erythromycin ribosomal methylase gene erm(B) and website https:// pubml st. org/. mef(A)-msr(D), while erm(B) alone was detected in Identification of acquired resistance was performed only two isolates. Isolates carrying  the erm(B) gene using the web-based platform ResFinder v3.2 of the had increased MIC for both erythromycin and azithro- Center for Genomic Epidemiology (http:// www. genom mycin, > 256  µg/mL, compared to isolates lacking  the icepi demio logy. org/). erm(B) gene (MIC 4-12 µg/mL), p < 0.001. For identification of mobile genetic elements and their The mef(A)-msr(D) gene predicted phenotypic resist- related acquired antimicrobial resistance we used the ance to macrolides, but at low MIC values (4–12 µg/mL Center for Genomic Epidemiology MobileElement Finder for erythromycin) and (6–96  µg/mL for azithromycin). v1.0.3 (http:// www. genom icepi demio logy. org/). Carrying the mef(A)-msr(D) gene alone did not predict This Whole Genome Shotgun project has been depos - phenotypic resistance to lincosamides (clindamycin) ited at DDBJ/ENA/GenBank under the BioProject num- and the six isolates carrying mef(A)-msr(D) were all ber PRJNA918594. phenotypically susceptible to clindamycin. Disc diffusion results showed that 13/26 (50%) of Statistical analysis PNSP isolates were resistant to tetracyclines. All the Categorical variables were presented in frequencies, per- 13-tetracycline resistant PNSP isolates carried the centages, and proportions. Categorical variables were tet(M) gene which confers resistance to tetracyclines. compared using chi square test. A p-value < 0.05 was con- Three PNSP isolates harbored cat (pC194) which con - sidered as threshold for statistical significance. Statistical fers resistance to chloramphenicol. analysis was performed using STATA version 16 (College The Tn6009-like element was detected in 13 PNSP. Station, TX). All tetracycline resistant PNSP were associated with a Tn6009 like element, while 12/13 of the erythromycin Results resistant PNSP had a Tn6009 like element. Twelve out A total of 26 penicillin non-susceptible Streptococcus of 13 tetracycline-resistant PNSP isolates were associ- pneumoniae were analyzed by whole genome sequenc- ated with plasmid replicon type repUS43. ing. Resistance to both macrolides and tetracyclines was Serotype 3 was the  most common, followed by sero- observed in 12/26 (46%) of PNSP isolates. type19  and 35B.  The majority of serotype  3 and 19 Phenotypic results using both EUCAST and CLSI PNSP displayed high level macrolide resistance and breakpoint interpretation showed that 13/26 (50%) of carried erm(B) and tet(M) genes. MLST analysis iden- PNSP isolates were resistant to macrolides (erythro- tified seventeen different sequence types (ST), among mycin). Phenotypic resistance to erythromycin was in which  ST271 accounted for 15% (4/26), followed by concordance with genotypic resistance determinants as ST172 (12%, 3/26) and ST14821 (8%, 2/26). The ST271 shown in Table 1. isolate  belonged to serotype 3 and carried multiple resistance-determinant genes. Manyahi et al. Ann Clin Microbiol Antimicrob (2023) 22:16 Page 4 of 7 Table 1 Phenotypic and Genotypic characteristics of penicillin non-susceptible Streptococcus pneumoniae Strain number Isolation Year Serotype ST PEN AZT ERY MLS genes TET Disc TET Plasmid Tn CHL MIC MIC MIC diffusion genes 2002c 2019 19F 5339 0.25 1.5 0.19 – – S – – – – 2145c 2019 34 5258 0.032 2 0.19 – – – S – – – 258d 2019 35B 6b3a 0.125 96 8 – mef(A) Msr(D) R Tet(M) repUS43 6009 – 469c 2018 35B 172 0.38 32 12 – mef(A) Msr(D) S – – – – 2010a 2018 19A 847 0.50 256 256 erm(B) – R Tet(M) repUS43 6009 - 263c 2018 6A 3460 0.125 1 0.125 – – – S – – – – 57c 2017 3 271 1.5 256 256 erm(B) mef(A) Msr(D) R Tet(M) repUS43 6009 – 2267a 2018 3 271 0.25 256 256 erm(B) mef(A) Msr(D) R Tet(M) repUS43 6009 – 90d 2018 15A 0c86 0.25 1 0.094 – – – S – – – 498d 2018 11A 14821 0.25 6 4 mef(A) Msr(D) R Tet(M) repUS43 6009 Cat(pC194) 116d 2018 11A 14821 0.19 32 4 mef(A) msr(D) R tet(M) repUS43 6009 Cat(pC194) 380a 2017 38 6103 0.25 1 0.094 – – – S – – – – 2071a 2018 3 2054 0.25 0.5 0.064 – – – S – rep36 – – 2019b 2018 4 9a19 0.50 1 0.125 – – – S – – – – 2267b 2018 3 271 0.75 256 256 erm(B) mef(A) msr(D) R tet(M) repUS43 6009 – 2052d 2019 3 700 0.38 0.75 0.094 – – – R tet(M) repUS43 6009 219a 2017 35B 172 0.25 1 0.094 – – – S – – – – 268a 2017 23B 6fe5 0.25 0.75 0.125 – – – S – – – – 2034b 2018 3 271 0.38 256 256 erm(B) mef(A) msr(D) R tet(M) repUS43 6009 393a 2017 35B 172 0.25 1 0.094 – – – S – – – – 2164a 2018 19F cf17 0.25 32 6 – mef(A) msr(D) R tet(M) rep13 6009 Cat(pC194) 272b 2017 19A 0c11 0.25 256 256 erm(B) – – R tet(M) repUS43 6009 – 258b 2017 21 cdc8 0.047 1 0.125 – – – S – – – – 252d 2017 46 15772 0.016 24 8 – mef(A) msr(D) R tet(M) repUS43 6009 – 2014d 2019 19F 8678 0.75 256 256 erm(B) mef(A) msr(D) R tet(M) repUS43 6009 – 369b 2018 23F 1188 0.25 2 0.125 – – – S – – – erm(B) Macrolide, Lincosamide and Streptogramin B resistance, mef(A) Macrolide resistance, msr(D) Macrolide resistance, tet(M) Tetracycline resistance, cat(pC194) Chloramphenicol resistance, AZT Azithromycin, ERY Erythromycin, PEN Penicillin, TET Tetracycline, CHL Chloramphenicol, ST sequence typing, MIC minimum inhibitory concentration M anyahi et al. Ann Clin Microbiol Antimicrob (2023) 22:16 Page 5 of 7 Discussion The erm(B) gene has been reported as the most com- We observed a discrepancy in azithromycin susceptibility mon macrolide resistance determinant in Streptococcus depending on whether using breakpoints from EUCAST pneumoniae in studies from Africa [14, 21, 22] and some or CLSI guidelines for interpretation. All 13 PNSP iso- part of Asia [11]. Macrolide resistance genes in Strepto- lates harboring genetic determinants for macrolide coccus pneumoniae have marked geographical variability resistance were correctly identified as resistant to eryth - [13]. The mef(A) has been reported to be the predomi - romycin and azithromycin (all with MIC ≥ 4  µg/mL) nant mechanism of pneumococcal macrolide resistance regardless of which breakpoints were used (sensitivity in North America and some parts of Europe [13]. Our 100%, 13/13). Using the EUCAST breakpoints appeared study found the macrolide efflux genes mef(A)/msr(D) to to overestimate azithromycin resistance, as all 13 eryth- be more prevalent (11/13) than the erm(B) genes (7/13). romycin-susceptible PNSP without genetic determinants Still, considering the high number of isolates harbor- of macrolide resistance were interpreted as resistant to ing both types of resistance genes (5/13), the dominant azithromycin (MIC-values from 0.5 to 2, specificity 0%, MLS phenotype was more frequent (7/13, erm(B) with 0/13). Using CLSI breakpoints only misclassified two or without mef(A)/msr(D)) than the M-phenotype (6/13, such isolates (MIC 2  µg/mL, specificity 85%, 11/13). mef(A)/msr(D) alone). Therefore, relying on current EUCAST guidelines Previous studies have shown that tetracycline and mac- appears to overestimate azithromycin resistance and rolide resistance genes are carried on mobile genetic ele- could in the clinical perspective lead to unnecessary use ments, composite conjugative transposons, Tn916-like of more broad-spectrum antibiotics. Our findings sug - elements, which facilitate their dissemination between gest that the EUCAST guidelines currently use a too different bacteria [23, 24]. Tn916 and Tn917-like com- low cutoff for MIC-values for azithromycin resistance in posite elements have been documented to facilitate dis- pneumococci. semination of erm(B) and mef(A/E) in Streptococcus PNSP susceptibility to erythromycin, on the other pneumoniae [17]. However, our study found a Tn6009- hand, was similar using both EUCAST and CLSI break- like element in all 13 PNSP isolates carrying the tet(M) points, and the phenotypic findings correlated well with gene which confers resistance to tetracycline, and in the identified genotypic resistance markers. To avoid var - 12/13 (92%) of PNSP isolates carrying macrolide resist- iations in interpretation, our findings call for AST guide - ance determinants. Tn6009 is a member of the Tn916– lines to be harmonized. Both CLSI and EUCAST state Tn1545  transposon family previously detected in that erythromycin susceptibility can predict susceptibil- Gram-positive and Gram-negative bacteria [25]. Tn6009 ity  to clarithromycin, azithromycin, dirithromycin, and has been reported to carry genes conferring resistance roxithromycin [19, 20]. Because almost all PNSP resistant against tetracycline tet(M), and inorganic and organic to erythromycin carried genetic determinants for mac- mercury [25]. Through horizontal gene transfer, the con - rolide resistance, our study supports that erythromycin jugative mobile elements enable bacteria to acquire and determines susceptibility to other macrolides. disseminate DNA between related and unrelated bacte- In Tanzania, macrolides are commonly used to treat ria. The presence of transposons containing macrolide respiratory tract infections. In the treatment of commu- and tetracycline resistance genes in PNSP in our study nity-acquired pneumonia, erythromycin and azithro- could indicate an increased risk of dissemination of these mycin are used as first and second line treatment, resistance determinants. respectively [6]. In this study, however, we observed that 50% and 58% of PNSP were resistant to erythromycin and azithromycin, respectively. Consequently, macrolides Conclusion appear potentially ineffective for treating PNSP infec - Macrolides and tetracyclines have only about 50% chance tions in this setting. This calls for prudent use of antibi - of being effective against PNSPs recovered from naso - otics including the use of narrow-spectrum penicillin. pharynx from people living with HIV in Dar es Salaam. But for treatment failure or infections likely caused by The erm(B) and mef(A)-msr(D) were common genes con- resistant pneumococci/PNSP,  options are difficult, with ferring resistance to macrolides and clindamycin, while azithromycin, the currently preferred treatment covering resistance to tetracycline was conferred by  the tet(M) just half of the PNSP. gene. Detection of the  composite conjugate transpo- The most common phenotype was MLS and isolates son Tn6009 associated with macrolides and tetracycline with this phenotype harbored the erm(B) gene confer- genes could indicate the possibility of horizontal transfer ring a high level of resistance to macrolides (> 256  µg/ of resistant genes. Using EUCAST guidelines for inter- mL) and clindamycin. All but two isolates with the ML S pretation overestimates azithromycin resistance in PNSP phenotype carried the mef(A) and msr(D) genes as well. compared to genetic correlates of resistance. Manyahi et al. Ann Clin Microbiol Antimicrob (2023) 22:16 Page 6 of 7 Acknowledgements a systematic analysis for the Global Burden of Disease Study 2016. Authors thank members of the bacteriology research laboratory of the Lancet Infect Dis. 2018;18(11):1191–210. https:// doi. org/ 10. 1016/ Muhimbili University of Health and Allied Science in Dar es Salaam, Tanzania, s1473- 3099(18) 30310-4. for their assistance during data collection and preliminary laboratory 4. Klugman KP. Pneumococcal resistance to antibiotics. Clin Microbiol procedures. Rev. 1990;3(2):171–96. https:// doi. org/ 10. 1128/ cmr.3. 2. 171. 5. Tleyjeh IM, Tlaygeh HM, Hejal R, Montori VM, Baddour LM. The impact Author contributions of penicillin resistance on short-term mortality in hospitalized adults BB and NL conceived the study. JM collected study data. JM and SM per- with pneumococcal pneumonia: a systematic review and meta-analy- formed the microbiological investigations. BB and JM performed statistical sis. Clin Infect Dis. 2006;42(6):788–97. https:// doi. org/ 10. 1086/ 500140. analysis. JM drafted the manuscript. BB, SJM, and NL revised the manuscript. 6. Tanzania standard treatment guidelines & national essential medicines All authors approved the final version. list. STG/NEMLIT 2021. Ministry of Health, Dar es Salaam, Tanzania 7. Mudhune S, Wamae M. Report on invasive disease and meningitis due to Funding Haemophilus influenzae and Streptococcus pneumonia from the Network This research was supported by (i) Helse Bergen HF, Haukeland University for Surveillance of Pneumococcal Disease in the East African Region. Clin Hospital, Bergen, Norway, project number 912132, , (ii) National Advisory Unit Infect Dis. 2009;48(Suppl 2):S147–52. https:// doi. org/ 10. 1086/ 596494. on Tropical Infectious Diseases, Haukeland University Hospital, Bergen, Nor- 8. Iroh Tam PY, Thielen BK, Obaro SK, Brearley AM, Kaizer AM, Chu H, et al. way, (iii) CAMRIA - Combatting Anti-Microbial Resistance with Interdisciplinary Childhood pneumococcal disease in Africa—a systematic review and Approaches, Centre for Antimicrobial Resistance in Western Norway, funded meta-analysis of incidence, serotype distribution, and antimicrobial sus- by Trond Mohn Stiftelse, grant number TMS2020TMT11, and (iv) STRESST - ceptibility. Vaccine. 2017;35(15):1817–27. https:// doi. org/ 10. 1016/j. vacci Antimicrobial Stewardship in Hospitals, Resistance Selection and Transfer in a ne. 2017. 02. 045. One Health Context, Liverpool School of Tropical Medicine, Liverpool, United 9. Sader HS, Mendes RE, Le J, Denys G, Flamm RK, Jones RN. Antimicrobial Kingdom, funded by JPIAMR grant number NFR333432. Susceptibility of Streptococcus pneumoniae from North America, Europe, Latin America, and the Asia-Pacific Region: results from 20 years of the Availability of data and materials SENTRY antimicrobial surveillance program (1997–2016). Open Forum Data are available on reasonable request. Infect Dis. 2019;6(Suppl 1):S14-s23. https:// doi. org/ 10. 1093/ ofid/ ofy263. 10. Yu YY, Xie XH, Ren L, Deng Y, Gao Y, Zhang Y, et al. Epidemiological char- acteristics of nasopharyngeal Streptococcus pneumoniae strains among Declarations children with pneumonia in Chongqing, China. Sci Rep. 2019;9(1):3324. https:// doi. org/ 10. 1038/ s41598- 019- 40088-6. Ethics approval and consent to participate 11. Beheshti M, Jabalameli F, Feizabadi MM, Hahsemi FB, Beigverdi R, Ethical approval to conduct the study were obtained from Muhimbili Univer- Emaneini M. Molecular characterization, antibiotic resistance pattern and sity of Heath and Allied Sciences Senate Research and Publications Committee capsular types of invasive Streptococcus pneumoniae isolated from clinical (Ref. No. 2015-10-27/AEC/Vol.X/54), National Health Research Ethics Commit- samples in Tehran, Iran. BMC Microbiol. 2020;20(1):167. https:// doi. org/ 10. tee (Ref. No. NIMRlHQ/R. SaJVol. 1X12144), Tanzania Medicines and Medical 1186/ s12866- 020- 01855-y. Devices Authority (Ref. No. TZ16CT007), and Regional Committee for Medical 12. Schroeder MR, Lohsen S, Chancey ST, Stephens DS. High-Level macrolide and Health Research Ethics of Western Norway (Ref. No. REK2015/540). resistance due to the mega element [mef(E)/mel] in Streptococcus pneu- Written informed consent was obtained from each study participant before moniae. Front Microbiol. 2019;10:868. https:// doi. org/ 10. 3389/ fmicb. 2019. the enrollment in the study. 13. Schroeder MR, Stephens DS. Macrolide resistance in Streptococcus pneu- Consent for publication moniae. Front Cell Infect Microbiol. 2016;6:98. https:// doi. org/ 10. 3389/ Consent to publish was obtained from National Health Research Ethics Com- fcimb. 2016. 00098. mittee (Ref. No. NIMRlHQ/R. SaJVol. 1X12144). 14. Raddaoui A, Tanfous FB, Chebbi Y, Achour W, Baaboura R, Benhassen A. High prevalence of multidrug-resistant international clones among Competing interests macrolide-resistant Streptococcus pneumoniae isolates in immunocom- Authors declare that they have no competing interests. promised patients in Tunisia. Int J Antimicrob Agents. 2018;52(6):893–7. https:// doi. org/ 10. 1016/j. ijant imicag. 2018. 04. 015. Author details 15. Korona-Glowniak I, Siwiec R, Malm A. Resistance determinants and their Department of Clinical Science, University of Bergen, Bergen, Norway. association with different transposons in the antibiotic-resistant Strepto - National Advisory Unit for Tropical Infectious Diseases, Department of Medi- coccus pneumoniae. Biomed Res Int. 2015;2015:836496. https:// doi. org/ cine, Haukeland University Hospital, Bergen, Norway. Department of Micro- 10. 1155/ 2015/ 836496. biology and Immunology, Muhimbili University of Health and Allied Sciences, 16. Talebi M, Azadegan A, Sadeghi J, Ahmadi A, Ghanei M, Katouli M, et al. P.O. Box 65001, Dar es Salaam, Tanzania. Determination of characteristics of erythromycin resistant Strep- tococcus pneumoniae with preferred PCV usage in Iran. PLoS ONE. Received: 5 September 2022 Accepted: 9 February 2023 2016;11(12):e0167803. https:// doi. org/ 10. 1371/ journ al. pone. 01678 03. 17. Chancey ST, Agrawal S, Schroeder MR, Farley MM, Tettelin H, Stephens DS. Composite mobile genetic elements disseminating macrolide resistance in Streptococcus pneumoniae. Front Microbiol. 2015;6:26. https:// doi. org/ 10. 3389/ fmicb. 2015. 00026. References 18. Manyahi J, Moyo S, Aboud S, Langeland N, Blomberg B. High rate of 1. Madhi SA, Petersen K, Madhi A, Khoosal M, Klugman KP. Increased antimicrobial resistance and multiple mutations in the dihydrofolate disease burden and antibiotic resistance of bacteria causing severe reductase gene among Streptococcus pneumoniae isolated from HIV- community-acquired lower respiratory tract infections in human infected adults in a community setting in Tanzania. J Glob Antimicrob immunodeficiency virus type 1-infected children. Clin Infect Dis. Resist. 2020;22:749–53. https:// doi. org/ 10. 1016/j. jgar. 2020. 06. 026. 2000;31(1):170–6. https:// doi. org/ 10. 1086/ 313925. 19. Clinical and Laboratory Standards Institute. Performance standards for 2. Wahl B, O’Brien KL, Greenbaum A, Majumder A, Liu L, Chu Y, et al. antimicrobial susceptibility testing; twenty-eighth informational sup- Burden of Streptococcus pneumoniae and Haemophilus influenzae plement CLSI document M100–S20. Wayne, PA: Clinical and Laboratory type b disease in children in the era of conjugate vaccines: global, Standards Institute; 2018. regional, and national estimates for 2000–15. Lancet Glob Health. 20. The European Committee on Antimicrobial Susceptibility Testing. Break- 2018;6(7):e744–57. https:// doi. org/ 10. 1016/ s2214- 109x(18) 30247-x. point tables for interpretation of MICs and zone diameters. Version 11.0, 3. Estimates of the global, regional, and national morbidity, mortality, and 2021. http:// www. eucast. org. aetiologies of lower respiratory infections in 195 countries, 1990–2016: M anyahi et al. Ann Clin Microbiol Antimicrob (2023) 22:16 Page 7 of 7 21. Midouni Ayadi B, Mehiri E, Draoui H, Ghariani A, Essalah L, Raoult D, et al. Phenotypic and molecular characterization of macrolide resistance mechanisms among Streptococcus pneumoniae isolated in Tunisia. J Med Microbiol. 2020;69(4):505–20. https:// doi. org/ 10. 1099/ jmm.0. 001151. 22. Diawara I, Zerouali K, Katfy K, Barguigua A, Belabbes H, Timinouni M, et al. Phenotypic and genotypic characterization of Streptococcus pneumo- niae resistant to macrolide in Casablanca, Morocco. Infect Genet Evol. 2016;40:200–4. https:// doi. org/ 10. 1016/j. meegid. 2016. 03. 003. 23. Roberts AP, Mullany P. Tn916-like genetic elements: a diverse group of modular mobile elements conferring antibiotic resistance. FEMS Micro- biol Rev. 2011;35(5):856–71. https:// doi. org/ 10. 1111/j. 1574- 6976. 2011. 00283.x. 24. Sansevere EA, Robinson DA. Staphylococci on ICE: overlooked agents of horizontal gene transfer. Mob Genet Elements. 2017;7(4):1–10. https:// doi. org/ 10. 1080/ 21592 56x. 2017. 13684 33. 25. Soge OO, Beck NK, White TM, No DB, Roberts MC. A novel transposon, Tn6009, composed of a Tn916 element linked with a Staphylococcus aureus mer operon. J Antimicrob Chemother. 2008;62(4):674–80. https:// doi. org/ 10. 1093/ jac/ dkn255. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Re Read ady y to to submit y submit your our re researc search h ? Choose BMC and benefit fr ? Choose BMC and benefit from om: : fast, convenient online submission thorough peer review by experienced researchers in your field rapid publication on acceptance support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year At BMC, research is always in progress. Learn more biomedcentral.com/submissions

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