Molecular Characterization of Penicillin-Binding Protein2x, 2b and 1a of Streptococcus pneumoniae Causing Invasive Pneumococcal Diseases in China: A Multicenter Study

Abstract

Streptococcus pneumoniae is a common human pathogen that can cause severe invasive pneumococcal diseases (IPDs). Penicillin-binding proteins (PBPs) are the targets for β-lactam antibiotics (BLAs), which are the common empirical drugs for treatment of pneumococcal infection. This study investigated the serotype distribution and antibiotic resistance patterns of S. pneumoniae strains causing IPD in China, including exploring the association between penicillin (PEN) susceptibility and PBPs variations. A total of 300 invasive S. pneumoniae isolates were collected from 27 teaching hospitals in China (2010-2015). Serotypes were determined by Quellung reaction. Serotypes 23F and 19F were the commonest serotypes in isolates from cerebrospinal fluid (CSF), whilst serotypes 19A and 23F were most commonly seen in non-CSF specimens. Among the 300 invasive S. pneumoniae strains, only one strain (serotype 6A, MIC = 0.25 μg/ml) with PEN MIC value ≤ 0.25 μg/ml did not have any substitutions in the PBPs active sites. All the strains with PEN MIC value ≥ 0.5 μg/ml had different substitutions within PBPs active sites. Substitutions in PBP2b and PBP2x active sites were common in low-level penicillin-resistant S. pneumoniae (PRSP) strains (MIC = 0.5 μg/ml), with or without PBP1a substitution, while all strains with PEN MIC ≥ 1 μg/ml had substitutions in PBP1a active sites, accompanied by PBP2b and PBP2x active site substitutions. Based on the three PBPs substitution combinations, a high degree of diversity was observed amongst the isolates. This study provides some new insights for understanding the serology and antibiotic resistance dynamics of S. pneumoniae causing IPD in China. However, further genomic studies are needed to facilitate a comprehensive understanding of antibiotic resistance mechanisms of S. pneumoniae.

Introduction

Streptococcus pneumoniae is one of the most common Gram-positive cocci that is mainly transmitted through the respiratory tract. The organism usually colonizes the human nasopharynx and can migrate to the middle ear and lungs causing local non-invasive pneumococcal disease (NIPD) such as otitis media and pneumonia in immune-deficient people (Lynch and Zhanel, 2009; Henriques-Normark and Tuomanen, 2013). Data from the World Health Organization (WHO) shows that pneumonia killed 808, 694 children under five years old in 2017, accounting for 15% of all deaths in children. S. pneumoniae is the most common pneumonia pathogen in children worldwide, with a mortality rate in children much higher than other diseases such as AIDS, malaria, and measles (Lynch and Zhanel, 2009). In Europe and America, S. pneumoniae is also the most common cause of community-acquired pneumonia in adults (World Health Organization [WHO], 2007). In addition to respiratory tract infections, S. pneumoniae can also migrate to the blood and brain and cause severe invasive pneumococcal disease (IPD), such as bacteremia, meningitis etc., causing a huge economic and medical burden on both developed and developing countries (Mehr and Wood, 2012).

The major empirical antimicrobial drugs used in the treatment of S. pneumoniae infections are β-lactam antibiotics (BLAs), which act on the bacterial cell wall. Penicillin-binding proteins (PBPs) are crucial enzymes in the biosynthesis of peptidoglycan (PG), a major cell wall component that surrounds the cytoplasmic membrane and is required to maintain the shape and osmotic stability of bacteria (Hakenbeck et al., 2012). The target of BLAs are PBPs, and function by covalently binding to the active site serine of PBPs through the β-lactam ring, thereby interfering with the synthesis of bacterial cell walls and eventually leading to bacterial cell death. With the widespread use of antibiotics, penicillin-intermediate S. pneumoniae (PISP) and penicillin-resistant S. pneumoniae (PRSP), commonly referred to as penicillin-non-susceptible S. pneumoniae (PNSP), have emerged and are detected continually worldwide. Data from the Asian Network for Surveillance of Resistant Pathogens (ANSORP) shows that the isolation rate of PNSP from 2012 to 2017 (9.0%) was significantly higher than that from 2008 to 2009 (4.9%), and the detection rates of PNSP from patients in China was 1.9% (2012-2017, oral breakpoint) (Kim et al., 2012, 2020).

The main mechanism of BLAs resistance by S. pneumoniae is through PBPs substitutions. Alterations in PBPs via substitutions reduce their reactivity for β-lactam attachment to the binding site and thereby reduce their effectiveness (Zapun et al., 2008). S. pneumoniae has six PBPs, but only three PBPs, PBP2x, PBP2b and PBP1a play a main role in BLAs resistance. Alterations in all other PBPs have been described occasionally (Zapun et al., 2008). Mosaicity is the product of homologous recombination that causes the sequence diversity in S. pneumoniae. In most resistant clinical S. pneumoniae isolates, the sequencing revealed that the mosaic genes encode PBP2x, PBP2b, and PBP1a (Laible et al., 1991; Martin et al., 1992; Sibold et al., 1994). The active sites of PBPs comprises three conserved sequences SXXK, SXN and KT(S)G. The serine of the SXXK motif is the active site residue that reacts with BLAs. They are located in PBP2x: 337STMK340, 395SSN397, 547KTG549; in PBP2b: 386STMK389, 443SSN445, and 615KTG617; and in PBP1a: 370STMK373, 428SRN430, 557KTG559 (Hakenbeck et al., 2012). Changes in these active site motifs and their adjacent sequences lead to a decrease in the affinity of PBPs to penicillin resulting in antibiotic resistance (Zhanel et al., 2006; Hakenbeck et al., 2012). Few studies have been carried out to understand the association between penicillin susceptibility and PBPs variations in S. pneumoniae isolates from patients with IPD in mainland China.

Due to the widespread use of antimicrobial drugs, and the fastidious nature of S. pneumoniae, the number of isolates from IPD specimens is very low, and hence a scarcity of relevant research studies (Xue et al., 2010; Yao et al., 2011; Quan-Cheng et al., 2016). This study aimed to analyze the serotype distribution and antibiotic resistance pattern of S. pneumoniae strains causing IPD in China, and to explore the association between penicillin susceptibility and PBPs variations.

Materials and Methods

Bacterial Isolates

A total of 300 non-duplicate invasive S. pneumoniae isolates from 27 teaching hospitals in 13 provinces of China (2010-2015) were studied (Supplementary Table 1). The isolates were transported to the Department of Clinical Laboratory in Peking Union Medical College Hospital for re-identification and further analysis. The most common specimen type was blood, accounting for 72.7% (218/300) of the isolates, followed by cerebrospinal fluid (CSF) (19.0%, 57/300) and pleural effusion (5.7%, 17/300). Other specimen types included ascites, joint drainages, pleural drainage and lung tissue, each accounting for ≤ 2.0% of the isolates. The majority of the patients were males, accounting for 65.7% (197/300) of the isolates. The average age of patients was 46 ± 26.69 years old.

Serotyping

All the S. pneumoniae isolates were serotyped by Quellung reaction (Maha et al., 2014). The serotype was first determined by latex agglutination test using the checkerboard typing system. Then specific type antiserum was mixed with the bacterial suspension to determine the final serotype. Capsular swelling was observed under oil immersion microscope. If the test strain was negative with all antisera, it was classified as non-typeable (NT).

Antimicrobial Susceptibility Testing

The minimum inhibitory concentrations (MICs) of S. pneumoniae against penicillin (PEN), amoxicillin/clavulanic (AMC), cefuroxime (CXM), ceftriaxone (CRO), cefepime (FEP), ertapenem (ETP), imipenem (IPM), meropenem (MEM), levofloxacin (LEV), trimethoprim/sulfamethoxazole (SXT), clindamycin (DA), clarithromycin (CLA), erythromycin (E), linezolid (LZD) and vancomycin (VA), were determined by broth microdilution method as recommended by the Clinical and Laboratory Standards Institute (CLSI) M07-A10 (Clinical and Laboratory Standards Institute (CLSI), 2012). S. pneumoniae ATCC49619 and Escherichia coli ATCC25922 were used as the quality control strains, and were tested along each batch. Results were considered valid when the MIC values of the quality control strains were within the expected range. Antimicrobial susceptibility testing results were interpreted according to CLSI 2019 guidelines (Clinical and Laboratory Standards Institute (CLSI), 2019).

Penicillin-Binding Protein Gene Amplification and Sequencing

The isolates were cultured on blood agar plates and incubated overnight at 35°C in a 5% CO₂ atmosphere. DNA was extracted using the AxyGen amp DNA Mini Extraction Kit (Axygen, United States) according to the manufacturer’s instructions. The final pure DNA was stored at −20°C until use. The nucleotide sequence of an around1-kb region encoding the penicillin-binding domain of pbp2x, pbp2b and pbp1a genes were amplified and sequenced based on published primers (Table 1; Zhanel et al., 2006). PCR products were sent to Beijing RuiBiotech Co., Ltd., for sequencing.

CLC Sequence Viewer software (CLC, Denmark) was used to manually correct the sequencing peaks and sequences to ensure the sequencing quality, and a two-way splicing was carried out. The spliced sequences were translated to simulated protein sequences and compared with PBP2x, PBP2b, and PBP1a corresponding to S. pneumoniae reference strain R6 (PSSP, GenBank accession No. NC003098) for variation analysis.

Data Analysis and Statistical Analysis

Differences in antimicrobial susceptibility were analyzed by MIC range, MIC₅₀ and MIC₉₀, and statistical analysis was performed by chi-square test or Fisher’s exact probability test using SPSS software (version 22.0, SPSS Inc., Chicago, IL, United States). P value < 0.05 was considered statistically significant.

Results

Serotype Distribution

Based on the Quellung reaction, a total of 299 S. pneumoniae isolates were identified to the serotype level accurately and one strain was considered as non-typeable (NT). Among the 299 serotypeable isolates, 41 serotypes were detected. The top five serotypes were: 23F (14.3%, 43/300), 19F (13.7%, 41/300), 19A (13.7%, 41/300), 3 (10.3%, 31/300) and 14 (9.0%, 27/300). The main serotypes in CSF isolates were 23F and 19F, both accounting for 15.8% (9/57) each, whilst in non-CSF specimens, serotypes 19A and 23F were the most common, accounting for 14.8% (36/243) and 14.0% (34/243) of the isolates, respectively (Table 2).

Antimicrobial Susceptibility

Concerning non-BLAs drugs, all the S. pneumoniae isolates were susceptible to LEV, VA and LZD, with MIC₉₀ values of 1 μg/ml, 0.5 μg/ml, and 1 μg/ml. The prevalence of resistance of the isolates to SXT was 65.3%. Resistance rates to CLA, E and DA were extremely high, all above 90%. PISP accounted for 4.3% of the isolates based on the non-meningitis (R ≥ 8 μg/ml) breakpoint while none, 67.7% and 44.7% of the isolates were classified as PRSP based on non-meningitis, meningitis (R ≥ 0.12 μg/ml) and oral administration (R ≥ 2 μg/ml) breakpoints, respectively. Susceptibility to AMC was about 97.7%. The prevalence of resistance of the isolates to the second-generation cephalosporins CXM was about ≥60%. The third- and fourth-generation cephalosporins CRO and FEP had the same MIC₉₀ value of 2 μg/ml. The resistance rates of the isolates to IPM and MEM was only 3.7 and 2.7%, respectively, but the intermediate rates were much higher at 37.7 and 46.0%, respectively (Table 3).

Association Between Serotypes, Penicillin Susceptibility and Variations in Penicillin-Binding Proteins Active Sites

Among the 300 isolates studied, 106 isolates, including all strains of serotypes1-5, 6C, 7F, 9A, 9N, 9V, 8, 17, 34, 10A, 11A, 12F, 15F, 17A, 18C, 24F, 25A, 25F, 28A, and 28F, one strain each of serotypes 6A, 7C, 19A and 23F, and two strains each of serotypes 6B, 13, 29, and 15A, exhibited PEN MIC values of ≤0.25 μg/ml. None of these isolates had PBPs substitution in the three conserved motifs and nearby sites, except one isolate of serotype 6A (PEN MIC = 0.25 μg/ml), in which TAA6A substitution was detected in the active sites of PBP2b. The remaining 194 isolates all had different amino acid substitutions in the PBPs active sites. In the PBP2x conserved motifs or nearby sites, 181 isolates had T338A substitution (threonine → alanine), 175 isolates had L546V substitution (leucine → valine), and 39 isolates had M339F substitution (methionine → phenylalanine), 4 isolates had H394L substitution (histidine → leucine), and the substitution rates were 60.3, 58.3, 13, and 1.3%, respectively. In the PBP2b conserved motifs or nearby sites, 190 isolates had T446A substitution (threonine → alanine), 3 isolates had T446S substitution (threonine → serine), and 72 isolates had A619G substitution (alanine → glycine), with substitution rates of 63.3, 1, and 24%, respectively. In the PBP1a conserved motifs or nearby sites, 53 isolates had T371A substitution (threonine → alanine), 122 isolates had T371S substitution (threonine → serine), and 175 isolates had P432T substitution (proline → threonine), with substitution rates of 17.7, 40.7, and 58.3%, respectively (Table 4).

Based on the serotype and PEN MIC distribution among the isolates, ten isolates had only one PBP gene active site or nearby sites substitution, including two isolates each of serotype 6A and 6B, one isolate each of serotypes 19F, 22 F and 23A, and three isolates of serotype 23F. Seven of these isolates had PBP2b active sites or nearby sites substitutions; two had only PBP2x active sites or nearby sites substitutions, while none had any substitution in single PBP1a active sites or nearby sites. Save for one isolate of serotype 6A with PEN MIC of 0.25 μg/ml, all these strains had an MIC of 0.5 μg/ml. Thirteen isolates had substitutions in two of the PBPs gene active sites or nearby sites, including four isolates of serotype 20, two isolates of serotype 23A, and one isolate each for serotypes 6B, 13, 14, 29, 15B, 19A, and 23F. Among them, three isolates had substitutions in both PBP2b and PBP1a genes (serotypes 19A, 23F, and 23A), and the rest had substitution in PBP2x and PBP2b. The PEN MIC of most isolates in the group with two substitutions in the PBPs gene active sites or nearby sites was 0.5 μg/ml. The remaining 171 isolates had substitutions in the active sites or nearby sites of all the three PBPs genes, mainly distributed in serotypes 14 (n = 26), 19A (n = 39), 19F (n = 40) and 23F (n = 38).

Taken together, all isolates of serotypes 1, 2, 3, 4, 5, 6C, 7F, 9A, 9N, 9V, 8, 17, 34, 10A, 11A, 12F, 15F, 17A, 18C, 24F, 25A, 25F, 28A, 28F, and 33B, had no amino acid changes in the three active sites or nearby sites of PBPs (MIC ≤ 0.25 μg/ml), and all isolates of serotypes 14, 15B, 15C, 19F, 22F, and 23A, had at least one substitution in the active sites or nearby sites of PBPs (0.5 μg/ml ≤ MIC ≤ 4 μg/ml).

Association Between Specimen Types and Variations in Penicillin-Binding Proteins Active Sites

Among the 300 invasive S. pneumoniae isolates, 57 (19.0%) were isolated from CSF, and the rest (81.0%) from other sterile body fluids, mostly in blood. Antimicrobial susceptibilities were interpreted according to meningitis and non-meningitis break points. PRSP and PSSP from CSF accounted for 70.2 and 29.8% of the isolates, respectively. PISP and PSSP from non-CSF accounted for 4.5 and 95.5% of the isolates, respectively. Although the proportion of PRSP derived from CSF was significantly higher than that from non-CSF sources (P < 0.0001), the proportion of CSF derived isolates with PEN MIC ≥ 1μg/ml (59.6%) was similar to that from non-CSF sources (54.3%) (P = 0.5644) (Figure 1).

FIGURE 1

Analysis of PBPs active sites of strains from CSF showed that all PSSP, and one strain of PRSP, with PEN MIC of 0.25 μg/ml, had no substitution in the PBPs active sites. PBP2x and PBP2b substitutions were common in low-level PRSP strains (MIC = 0.5μg/ml), with or without PBP1a substitution. The strains with PEN MIC ≥ 1 μg/ml all had substitutions in the PBP1a active site or nearby sites, accompanied by substitutions in the PBP2x and PBP2b regions (Table 5).

Analysis of PBPs active sites or nearby sites of strains derived from non-CSF sources revealed that all PSSP with PEN MIC ≤ 0.25 μg/ml had no substitutions in PBPs active sites or nearby sites, except one serotype 6A strain, in which a T446A substitution nearby the active site of PBP2b was detected. Various substitutions in the PBPs active sites or nearby sites of PSSP strains with PEN MIC ≥ 0.25 μg/ml were detected. Similar to CSF derived strains, the substitutions in active sites or nearby sites of PBP2x and PBP2b were common in strains with low PEN MIC level, with or without PBP1a active site substitution. In contrast, strains with PEN MIC ≥ 1 μg/ml all had substitutions in PBP1a active sites or nearby sites, accompanied by PBP2x or PBP2b substitutions (Table 6).

Variations of the pbp2x Gene

Compared with the reference strain R6 (GenBank accession No. NC003098), a total of 338 amino acids from positions 259 to 596 of the PBP2x protein were analyzed. A total of 98 different substitutions at 73 amino acid positions were detected in the PBP2x sequence, among which D567N (Asp → Asn) was the most common substitution, accounting for 69.3% (208/300), followed by D488N (Asp → Asn) and S576N (Ser → Asn), each accounting for 64.0% (192/300). Strains with L565S (Leu → Ser) and R384G (Arg → Gly) substitutions each accounted for 62.7% (188/300) and 61.3% (184/300), respectively. According to the distribution of various substitutions in PBP2x, all strains could be divided into 43 groups. The number of substitution sites in each group ranged from 1 to 42, accounting for 0-12.4% of the total number of amino acids analyzed. Group 2X37 was the commonest, accounting for 16.3% of all strains (49/300). There were 38 substitutions detected in all strains within the group, and the substitution rate was 11.2%. The second common group was 2X01, accounting for 13.3% of all strains (40/300), and the PBP2x sequence of strains in this group was exactly the same as that of R6 strain. There were 33 strains in the 2X41 group, accounting for 11.0% of the strains. Forty substitutions were detected in all strains of this group and the substitution rate was 11.8% (Table 7). Compared with PBP2b and PBP1a sequences, PBP2x had the least number of total substitutions (98 vs. 112 vs. 105). According to the distribution of PEN MIC, the number of detectable substitution sites in strains with PEN MIC ≤ 0.25 μg/ml ranged from 10-32, while for strains with PEN MIC = 0.5, 1, 2, and 4 μg/ml the number of detectable substitutions were 86, 46, 57, and 48, respectively (Supplementary Table 2).

Variations of the pbp2b Gene

Compared with the reference strain R6 (GenBank accession No. NC003098), a total of 372 amino acids from positions 305 to 676 of the PBP2b protein were analyzed for the isolates. A total of 111 different substitutions and two insertion variations at 88 amino acid positions were detected in the PBP2b sequence. The two insertion variations were only found in two strains, both located between amino acids 424-425, and the insertion sequences were YIW (Tyr–Ile-Try) and YTW (Tyr-Thr-Try). The most common substitution was E476G (Glu → Gly), accounting for 64.7% (194/300) of the isolates, followed by T446A (Thr → Ala), Q438E (Gln → Glu), L455I (Leu → Ile), which accounted for 63.3% (190/300), 61.7% (185/300) and 61.0% (183/300), respectively. According to the distribution of various substitutions in PBP2b, all strains could be divided into 46 groups. Except for group 2B45 and 2B46, each containing one insertion variation, the other groups all had amino acid substitutions in PBP2b protein. The number of substituted amino acid sites ranged from 0 to 45, accounting for 0-12.1% of the total number of amino acids (n = 372) analyzed. Group 2B01 was the most common, accounting for 17.3% of the strains (52/300), and had exactly the same sequence of PBP2b as reference strain R6. The second common group was 2B20, accounting for 14.3% of the strains (43/300). A total of 13 amino acid substitutions were detected in the PBP2b sequence of the strains in this group, and the substitution rate was 3.5% (Table 8). According to the distribution of PEN MICs, the number of substitution sites in strains with MIC ≤ 0.12μg/ml were less than ten, while the number of detectable substitution sites in strains with MIC = 0.25, 0.5, 1, 2, and 4μg/ml were 12, 75, 61, 52, and 54, respectively (Supplementary Table 3).