Russian strains of group B streptococci are different in the content and organization of the PAI-A and PAI-A1 pathogenicity islands

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BACKGROUND

Group B streptococci (GBS) were exclusively considered as causative agents of mastitis in cows for a long time, but later on, were discovered to cause various diseases in humans, affecting a variety of organs and tissues [1].

GBS infection in newborns can have an early (first 6 days of life) and late onset (7 days of life to 3 months). GBS can result in sepsis, pneumonia, and less often meningitis in early onset of infection, whereas late infection can cause meningitis. The GBS infection incidence in newborns accounts for 0.53 cases per 1000 live births, and the mortality rate among the affected is 9.6% [2, 3]. Neonatal diseases caused by GBS are widespread worldwide, and the incidence of GBS colonization in pregnant women ranges from 10% to 30% in the United States of America (USA), 6.5%–36% in Europe, 7.1%–16% in Asia, 9.1%–25.3% in the Middle East, and 11.9%–31.6% in Africa [3, 4].

GBS leads to invasive (bloodstream infections, meningitis, osteomyelitis, and endocarditis) and non-invasive infections (bacteriuria, fasciitis, cellulitis, endometritis, and wound infections) in pregnant women and young mothers [4].

In addition, GBS can cause arthritis, endocarditis, pneumonia, and bacteremia, as well as urinary tract, soft tissue, skin, and bone infections in adults. The risk group includes people with weakened immunity and the elderly [4].

Currently, ten serotypes of GBS are known (Ia, Ib, and II–IX). The prevalence and dominance of GBS serotypes vary by geographic region. Thus, serotypes Ia, Ib, II, III, and V prevail among the colonizing strains in the USA and Europe, VI and VIII prevail among the pregnant women in Japan, IV and V prevail among the pregnant women in the UAE and Egypt, respectively. Most cases of late onset meningitis in newborns are caused by serotype III invasive strains, whereas in other cases (not in pregnant women), invasive isolates of serotypes Ia and V dominate. However, recent studies showed that both invasive strains appeared in newborns and adults, as well as colonizing serotype IV strains. In Malaysia, serotype VI was found among the dominant colonizing strains and is the second most common isolate in adults with skin and soft tissue infections caused by GBS. In Egypt, serotype VI is a common colonizing serotype in women. Recently in Central Taiwan, the spread of serotype VI was revealed among colonizing and invasive isolates. Several studies identified sporadic serotype VIII strains both among the colonizing and causative agents of invasive GBS diseases. Serotype V was similarly identified in the USA and Europe [4].

The causative agents of 97% of invasive B-streptococcal infections of newborns in all geographic regions are GBS of five serotypes (Ia, Ib, II, III, and V), dominated by serotype III [5].

Most cases of diseases in adults caused by GBS are caused by serotypes V, Ia, and III [6].

Scientists are developing multicomponent vaccines that will protect against GBS of the predominant serotypes since the distribution of serotypes is different in geographic regions of the world.

The pharmaceutical company, GlaxoSmithKline (GSK), has completed phase I/II clinical trials of the CPS-CRM197 trivalent conjugate vaccine against GBS (serotypes Ia, Ib, and III) and is planning for a pentavalent vaccine clinical trials (serotypes Ia, Ib, II, III, and V) [6].

The pharmaceutical company, Pfizer, also announced a clinical trial of a pentavalent conjugate vaccine (serotypes Ia, Ib, II, III, and V) to assess the safety, tolerance, and immunogenicity [6].

The rapid adaptation of GBS to various ecological niches can be due to many factors of pathogenicity. Some of them are described below.

The Bac protein interacts with the components of the human immune system, such as Fc-fragment of immunoglobulin A and factor H [7].

Lipoprotein ScaAB is involved in the transport of divalent metals and the adhesion process [8].

Serine protease C5a-peptidase cleaves and inactivates the C5a component of human complement [9, 10] and binds fibronectin and promotes bacterial invasion of the epithelial cells [7].

CspA is another serine protease, which cleaves the fibronectin extracellular matrix [11].

The surface protein SspB1 includes á-helical structures, a glucan-binding domain, and an anchoring site in the LPXTG cell wall, which classify this protein as an adhesin. The SspB1a protein is its homolog; its identity at the protein level is 72% [12]. According to the authors, the presence of genes of the sspB family does not significantly affect the course of pregnancy but can induce intrauterine infection in the fetus and newborn [13]. These proteins can promote adhesion to the genitourinary tract epithelium. The presence of sspB genes correlates with urogenital tract infections [12].

Due to the variability of serotypes in different regions worldwide, creating a universal vaccine is impossible; therefore, the creation of recombinant vaccines based on surface proteins has been initiated.

Thus, the Department of Molecular Microbiology of the Institute of Experimental Medicine has developed pentavalent recombinant and recombinant chimeric vaccines (based on five surface proteins Bac, ScaAB, SspB1, ScpB, and CspA), as well as a live vaccine based on the probiotic strain Enterococcus faecium L3 [14–16].

Many factors of GBS pathogenicity are localized on mobile genetic elements; therefore, horizontal gene transfer can contribute to the formation of highly new virulent strains. At the beginning of the XXI century, 14–15 pathogenicity islands were found in GBS [17, 18].

Pathogenicity islands PAI-A and PAI-A1 containing the sspB1 and sspB1a genes, respectively, were identified among the mobile genetic elements of GBS [19–21].

The incidence of sspB1 and sspB1a genes, and therefore of the pathogenicity islands PAI-A and PAI-A1 in St. Petersburg, is 9% and 29%, respectively [21]. However, analysis of the National Center for Biotechnology Information databases indicates a significantly rarer incidence of the PAI-A island worldwide. Information on the prevalence of the patho­genicity islands under study among GBS of various serotypes in other regions of Russia is unavailable. Evidence is also unavailable in the relationship between the presence of these pathogenicity islands in the genome and the virulence of GBS strains.

Therefore, this study aimed to determine the incidence of pathogenicity islands, PAI-A and PAI-A1, among clinical and colonizing strains of GBS.

MATERIALS AND METHODS

Bacterial strains and cultivation conditions

This study included the following strains:

1. 24 strains isolated at the V.I. Kulakov National Medical Research Center for Obstetrics, Gynecology, and Perinatology in 2014–2016 (clinical);
2. 43 strains were obtained from the I.M. Sechenov First Moscow State Medical University (Sechenov University) in 1987–1990 (colonizing).

The GBS strains were grown in Todd Hewitt Broth (Pronadisa, Spain) overnight at 37°C.

Polymerase chain reaction (PCR)

Deoxyribonucleic acid (DNA) from GBS strains was isolated using the Express-DNA-Bio kit (Alkor Bio, Russia) according to the manufacturer’s recommendations.

Amplification was performed in a C1000 apparatus (BioRad, USA) using a two-fold Dream-Taq Green master mix (Thermo Fisher Scientific, Lithuania) according to the manufacturer’s recommendations and primers presented in Table 1.

 

Table 1. Primers used to identify the sspB1 and sspB1a genes

Primer name	Primer sequence	Product size, bp	Gene
sspB1971	cgt gat att acg ttt ggc aag a	1536	Surface adhesin SspB1
sspB3485	cca gtt cct gaa ccg ata aaa g
sspB1aF	tgg taa tat tct ccc cct tgg	220	SspB1a
sspB1aR	ttg cca gat gaa gca gct att

 

PCR results were evaluated in 1% agarose gel supplemented with ethidium bromide using ×1 TAE buffer. The results were visualized in a VersaDoc transilluminator.

Multiplex PCR

Multiplex PCR was used to identify the type of GBS by the polysaccharide capsule nature [22].

Statistical analysis

The significance of differences between collections was assessed using Fisher’s exact test. Therefore, we used an online program at the website https://molbiol.kirov.ru/utilites/multitool/.

RESULTS AND DISCUSSION

Determination of serotypes in the studied strains of GBS

Multiplex PCR was conducted to determine the genes of the capsular operon from the DNA of the strains described above to investigate the prevalence of GBS serotypes in various population groups [22]. The serotype of one strain from the Kulakov Institute and 18 strains from the Sechenov University could not be determined by this method. The group of clinical strains also included the previously analyzed GBS strains isolated at the D.O. Ott Scientific Research Institute of Obstetrics, Gynecology, and Reproductology (173 strains) and the Mechnikov North-Western State Medical University (1 strain) [21, 23]. The results are presented in Table 2.

 

Table 2. Incidence of serotypes (%) in the studied strains of group B streptococci

Serotypes	Colonizing	Clinical
Ia	23	22
Ib	7	4
II	19*	8*
III	0*	18*
IV	0*	18*
V	2	12
VI	0	0
VII	2	1
VIII	5*	0*

* Statistically significant differences between the two groups (p < 0.05).

 

Among the colonizing strains, serotypes Ia, II, and Ib dominate, whereas, among the clinical ones, Ia, II, III, IV, and V serotypes are predominant. Serotypes VII and VIII, rare for Russia, were revealed in both collections.

Thus, serotype Ia predominates in both collections, serotypes II and VIII are more common in colonizing strains, whereas serotypes III and IV are characteristic only for strains isolated from pregnant women and newborns. ­Obtained results correspond to the literature data, which state that the predominant serotypes in all geographic regions are Ia, Ib, II, III, and V. The comparison of strains within the collection of clinical strains established that serotype IV is more typical for strains from St. Petersburg and serotype III is more characteristic for strains from Moscow.

Incidence of pathogenicity islands PAI-A and PAI-A1 of GBS associated with genes sspB1 and sspB1a

Two homologous islands of pathogenicity, PAI-A and PAI-A1, were revealed in the genome of GBS strains [21]. The structure of pathogenicity islands is presented in Figure 1.

 

Fig. 1. The structure of the PAI-A and PAI-A1 pathogenicity islands of group B Streptococci. Vertical lines indicate homologous areas

 

PCR was performed for the sspB1 and sspB1a genes, which are marker genes of the pathogenicity islands described above to determine the incidence of these mobile genetic elements. The sspB1 and sspB1a genes were previously established to localize in the genomes of 9% and 29% of strains from St. Petersburg, respectively [21]. Figure 2 presents the PCR results with the DNA of clinical strains for the sspB1a gene.

 

Fig. 2. Electropherogram of the polymerase chain reaction products with clinical strains for the sspB1a gene:  1 — strain 2978, 2 — strain 3093, 3 — strain 2828, 4 — strain 3481, 5 — strain 3482, 6 — strain 2698, 7 — strain 3085, 8 — strain 3030, 9 — positive control

 

The sspB1 gene was revealed in the genomes of 8% of clinical strains. However, strains with the sspB1 gene were absent in the strains from Moscow (Table 3). Consequently, strains of GBS with a PAI-A island of pathogenicity circulate mainly in St. Petersburg.

 

Table 3. Incidence of sspB1 and sspB1a genes (%) in the studied strains of group B streptococci

Genes	Colonizing	Clinical
sspB1	0	8
sspB1a	9*	30*

* statistically significant differences between the two groups (p < 0.05).

 

PCR results revealed that the sspB1a gene is found in the genomes of 30% of clinical strains and 9% of colo­nizing strains. The sspB1a gene is more often revealed in the genomes of clinical strains, as opposed to colonizing ones.

Thus, it can be assumed that the genes of the sspB family are more typical for GBS colonizing pregnant women and newborns.

CONCLUSION

The predominant serotypes among clinical and colonizing strains of GBS correspond to the prevalence data for serotypes worldwide.

The GBS strains with the PAI-A pathogenicity island in their genomes circulate mainly in St. Petersburg.

The homologous pathogenicity island PAI-A1 is characterized by a higher prevalence, as the incidence of this mobile genetic element is three times higher in clinical GBS strains compared to colonizing strains.

The higher prevalence of genes of the sspB family among the genomes of clinical strains is of great importance since the SspB1 protein is included in the vaccine against GBS. Since the homology of SspB1 and SspB1a proteins is 72%, the vaccine is possible to act on strains with the sspB1a gene.

ADDITIONAL INFORMATION

Conflict of interest. The authors declare no conflict of in­terest.

Funding. This study was supported by the budgetary funds of the Institute of Experimental Medicine (code 0557-20190002, registration number R&D AAAA-A19-119022290066-6).

About the authors

Evgenia V. Kuleshevich

Institute of Experimental Medicine

Email: k-zh-v@mail.ru
ORCID iD: 0000-0002-3180-6491

Cand. Sci. (Biol.)

Russian Federation, Saint Petersburg

Yury Y. Ilyasov

Institute of Experimental Medicine

Email: kolpino@hotmail.com
SPIN-code: 2204-1677
Russian Federation, Saint Petersburg

Dmitry S. Linnik

Institute of Experimental Medicine

Email: linnikdm@yandex.ru
ORCID iD: 0000-0002-8046-9743
Russian Federation, Saint Petersburg

Anastasia A. Malchenkova

Institute of Experimental Medicine

Email: nastya.malchenkova@yandex.ru
ORCID iD: 0000-0003-2201-0472
SPIN-code: 8957-5056
Russian Federation, Saint Petersburg

Olga N. Arzhanova

Research Institute of Obstetrics, Gynecology, and Reproductology named after D.O. Ott,
Saint Petersburg State University

Email: arjanova_olga@mail.ru
ORCID iD: 0000-0003-3059-9811

MD, Dr. Sci. (Med.), Professor, Honored Doctor of the Russian Federation, Leading Researcher. The Department of Obstetrics and Perinatology; Professor. The Department of Obstetrics, Gynecology, and Reproductive Sciences, Medical Faculty

Russian Federation, 3 Mendeleevskaya Line, Saint Petersburg, 199034; 7-9 Universitetskaya emb., St. Petersburg, 199034

Nikolay I. Briko

I.M. Sechenov First Moscow State Medical University

Email: nbrico@mail.ru
ORCID iD: 0000-0002-6446-2744
SPIN-code: 2992-6915

MD, Dr. Sci. (Med.), Professor, Honored Scientist of the Russian Federation, Academician of the Russian Academy of Sciences

Russian Federation, 8-2 Trubetskaya str., Moscow, 119991

Ekaterina V. Glushkova

I.M. Sechenov First Moscow State Medical University

Email: ekaterina-1801@mail.ru
ORCID iD: 0000-0001-6997-7598
SPIN-code: 2336-3176

MD, Cand. Sci. (Med.)

Russian Federation, 8-2 Trubetskaya str., Moscow, 119991

Tatyana V. Priputnevich

National Medical Research Center for Obstetrics, Gynecology and Perinatology named after Academician V.I. Kulakov

Email: priput1@gmail.com
ORCID iD: 0000-0002-4126-9730
SPIN-code: 8383-7023

MD, Dr. Sci. (Med.)

Russian Federation, 4 Street Akademika Oparina, Moscow, 117997

Alexander N. Suvorov

Institute of Experimental Medicine; Saint Petersburg State University

Author for correspondence.
Email: alexander_suvorov1@hotmail.com
ORCID iD: 0000-0003-2312-5589
SPIN-code: 8062-5281

MD, Dr. Sci. (Med.), Professor, Corresponding Member of the Russian Academy of Sciences

Russian Federation, Saint Petersburg; Saint Petersburg

References

Supplementary files