Manyahi et al.

Ann Clin Microbiol Antimicrob (2023) 22:16 Annals of Clinical Microbiology

https://doi.org/10.1186/s12941-023-00565-3
and Antimicrobials

RESEARCH Open Access

Genetic determinants of macrolide

and tetracycline resistance in penicillin
non‑susceptible Streptococcus pneumoniae
isolates from people living with HIV in Dar es
Salaam, Tanzania
Joel Manyahi3*, Sabrina J. Moyo1,2,3, Nina Langeland1,2 and Bjørn Blomberg1,2

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 M ­ LSB
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 ­MLSB in PNSP. Resistance to
tetracycline was conferred by the tet(M) gene. Resistance genes were associated with the Tn6009 transposon.

*Correspondence:
Joel Manyahi
[email protected]
Full list of author information is available at the end of the article

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Manyahi et al. Ann Clin Microbiol Antimicrob (2023) 22:16 Page 2 of 7

Background producing ­MLSB phenotypes [13, 14]. Macrolide efflux

Streptococcus pneumoniae is a transient colonizer of protein A and E, efflux pumps encoded by the mef(A) and
the nasopharynx, with colonization peaking in the early mef(E) genes, and ribosomal mutations (23S rRNA), are
years of life and declining into adulthood. It is associated other common causes of macrolide resistance in Strep-
with both invasive and non-invasive pneumococcal dis- tococcus pneumoniae [13]. The mef(A/E) genes confer
eases. The incidence of invasive pneumococcal diseases the M phenotype, exhibiting low level resistance to mac-
is most prominent at the extremes of age, as well as in rolides, but not resistance to lincosamides and strepto-
immunocompromised hosts and people with chronic gramin B. The macrolide resistance genes are commonly
respiratory tract diseases. People living with HIV are carried on mobile genetic elements, facilitating their easy
particularly vulnerable to severe pneumococcal disease intra- and interspecies dissemination [15, 16]. The Tn916
[1] Globally, Streptococcus pneumoniae is estimated to transposon family that contains the tetracycline resist-
have caused as many as 515,000 deaths (95% uncertainty ance determinant tet(M), has frequently been reported to
interval, UI 302,000–609,000) in children aged < 5 years harbor macrolide resistance determinant genes [17].
in 2015, and approximately 50% of those deaths occurred Macrolide resistance determinants vary with geograph-
in four countries in Africa (Nigeria, Democratic republic ical locations [13]. In Tanzania, where macrolides and
of Congo) and Asia (India, Pakistan) [2]. Globally, Strep- tetracyclines are commonly used and easily accessible
tococcus pneumoniae is estimated to cause 1,189,937 over the counter without prescriptions, the mechanisms
deaths (UI 690,445-1,770,660) [3]. of resistance to these antibiotics in Streptococcus pneu-
Before 1970, pneumococci were readily susceptible moniae has not been studied. Therefore, we performed
to nearly all relevant antibiotics, and penicillin was the this study using whole genome sequencing to determine
antibiotic of choice. In the late 1970s, pneumococci with mechanisms of antibiotic resistance among penicillin
non-susceptibility to penicillin emerged, resulting in non-susceptible Streptococcus pneumoniae isolated from
treatment failures [4, 5]. The discovery of pneumococci Tanzania.
resistant to penicillin shifted empirical treatment for
suspected bacterial respiratory tract infection to mac- Materials and methods
rolides and tetracycline. In Tanzania, standard treatment Bacterial isolates
guidelines recommend macrolides and tetracyclines as Twenty-six penicillin non-susceptible Streptococcus
first and second line treatments, respectively, for mild pneumoniae were isolated by culturing nasopharyngeal
to moderate community acquired pneumonia caused by swabs obtained from healthy HIV infected adults in Tan-
Streptococcus pneumoniae [6]. However, the recommen- zania as part of the randomized clinical trial CoTrim-
dation is not based on current evidence of susceptibility Resist (ClinicalTrials.gov identifier: NCT03087890,
patterns, as surveillance of the trend of antibiotic resist- registered on 23rd March, 2017). The study popula-
ance in Streptococcus pneumoniae is limited in Tanzania. tion and bacterial isolates have been described previ-
Data from the Network for Surveillance of Pneumococcal ously [18]. Streptococcus pneumoniae was identified by
Disease in the East African Region in the pre-pneumo- conventional methods including optochin disk and bile
coccal vaccination era reported a low rate of Streptococ- susceptibility and further confirmation was done by
cus pneumoniae resistant to erythromycin and other Matrix-Assisted Laser Desorption/Ionization-Time of
antibiotics in Tanzania [7]. Furthermore, a meta-analysis Flight (MALDI-TOF) mass spectrometry (MS), using the
of childhood pneumococcal diseases in Africa prior to Microflex LT instrument and MALDI Biotyper 3.1 soft-
the widespread use of the pneumococcal capsular vac- ware (Bruker Daltonics, Bremen, Germany). Isolates with
cine (PCV) reported a low rate of resistance to eryth- discordant results between MALDI-TOF and conven-
romycin, but a substantially higher rate of resistance to tional identification with optochin disk and bile suscepti-
tetracycline [8]. bility were omitted.

formed by latex agglutination (Immulex™ Pneumotest

However, post-PCV surveillance studies conducted in Serotyping of Streptococcus pneumoniae was per-
well-organized settings have shown increased pneumo-
coccal resistance to erythromycin and other antibiotics, Kit; SSI Diagnostica A/S, Hillerød, Denmark).
partly attributed to increased consumption of macrolides
[9–11]. Antimicrobial susceptibility testing
Pneumococcal resistance to macrolides is mediated by The E-test strip (bioMérieux, Marcy-I-Etoile, France) was
erythromycin ribosomal methylase B (erm(B)) encoding used to determine the minimum inhibitory concentra-
enzymes that methylate the 23S rRNA, thereby inhibit- tions (MIC) for azithromycin, erythromycin and peni-
ing macrolide binding [12]. The erm(B) confers resist- cillin. The disk diffusion method was used to determine
ance to macrolides, lincosamides, and Streptogramin B, tetracycline and clindamycin susceptibility [19]. Muller
Manyahi 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 ­ O2 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).
Whole genome sequencing and analysis Genes conferring resistance to the group of mac-
Whole genome sequencing was performed using the Next rolides, lincosamides, and streptogramin B were
generation sequencing platform HiSeq X10 (Illumina, observed in all the 13-erythromycin resistant PNSP iso-
San Diego, CA, USA) at MicrobesNG (Microbes NG, lates. The M­ LSB phenotype (resistance to macrolides,
Birmingham, UK). Quality filtering and sequencing short lincosamides, and streptogramin B) accounted for 54%
read trimming were performed by MicrobesNG using (7/13) while the M phenotype (resistance to macrolides,
SPAdes and annotated in GenBank. Short read sequences but not to lincosamides or streptogramin B) accounted
were assembled using Unicycler at MicrobesNG. for the remaining 46% (6/13).
For allocation of multi-locus sequence typing (MLST) The macrolide efflux gene mef(A)-msr(D) was
and clonal complex, we used the online MLST database observed in 6 isolates. Five isolates carried both the
website https://​pubml​st.​org/. erythromycin ribosomal methylase gene erm(B) and
Identification of acquired resistance was performed mef(A)-msr(D), while erm(B) alone was detected in
using the web-based platform ResFinder v3.2 of the only two isolates. Isolates carrying the erm(B) gene
Center for Genomic Epidemiology (http://​www.​genom​ had increased MIC for both erythromycin and azithro-
icepi​demio​logy.​org/). mycin, > 256 µg/mL, compared to isolates lacking the
For identification of mobile genetic elements and their erm(B) gene (MIC 4-12 µg/mL), p < 0.001.
related acquired antimicrobial resistance we used the The mef(A)-msr(D) gene predicted phenotypic resist-
Center for Genomic Epidemiology MobileElement Finder ance to macrolides, but at low MIC values (4–12 µg/mL
v1.0.3 (http://​www.​genom​icepi​demio​logy.​org/). for erythromycin) and (6–96 µg/mL for azithromycin).
This Whole Genome Shotgun project has been depos- Carrying the mef(A)-msr(D) gene alone did not predict
ited at DDBJ/ENA/GenBank under the BioProject num- phenotypic resistance to lincosamides (clindamycin)
ber PRJNA918594. and the six isolates carrying mef(A)-msr(D) were all
phenotypically susceptible to clindamycin.
Statistical analysis Disc diffusion results showed that 13/26 (50%) of
Categorical variables were presented in frequencies, per- PNSP isolates were resistant to tetracyclines. All the
centages, and proportions. Categorical variables were 13-tetracycline resistant PNSP isolates carried the
compared using chi square test. A p-value < 0.05 was con- tet(M) gene which confers resistance to tetracyclines.
sidered as threshold for statistical significance. Statistical Three PNSP isolates harbored cat (pC194) which con-
analysis was performed using STATA version 16 (College fers resistance to chloramphenicol.
Station, TX). The Tn6009-like element was detected in 13 PNSP.
All tetracycline resistant PNSP were associated with a
Results Tn6009 like element, while 12/13 of the erythromycin
A total of 26 penicillin non-susceptible Streptococcus resistant PNSP had a Tn6009 like element. Twelve out
pneumoniae were analyzed by whole genome sequenc- of 13 tetracycline-resistant PNSP isolates were associ-
ing. Resistance to both macrolides and tetracyclines was ated with plasmid replicon type repUS43.
observed in 12/26 (46%) of PNSP isolates. Serotype 3 was the most common, followed by sero-
Phenotypic results using both EUCAST and CLSI type19 and 35B. The majority of serotype 3 and 19
breakpoint interpretation showed that 13/26 (50%) of PNSP displayed high level macrolide resistance and
PNSP isolates were resistant to macrolides (erythro- carried erm(B) and tet(M) genes. MLST analysis iden-
mycin). Phenotypic resistance to erythromycin was in tified seventeen different sequence types (ST), among
concordance with genotypic resistance determinants as which ST271 accounted for 15% (4/26), followed by
shown in Table 1. ST172 (12%, 3/26) and ST14821 (8%, 2/26). The ST271
isolate belonged to serotype 3 and carried multiple
resistance-determinant genes.
Table 1 Phenotypic and Genotypic characteristics of penicillin non-susceptible Streptococcus pneumoniae
Strain number Isolation Year Serotype ST PEN AZT ERY MLSB 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 – – – –
Manyahi et al. Ann Clin Microbiol Antimicrob

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 –
(2023) 22:16

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
Page 4 of 7
Manyahi 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%, ­MLSB 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 ­MLSB 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 M ­ LSB 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

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