Serotyping and Antibiotic Susceptibility of Invasive Streptococcus agalactiae in Egyptian Patients with or without Diabetes Mellitus

ABSTRACT.

Streptococcus agalactiae serotype distribution and its antibiotic susceptibility affect disease prevention strategies, but the serotype distribution varies among patient groups. The objectives of this study were to establish the group B Streptococcus (GBS) serotype distribution in patients from Egypt and to assess antibiotic sensitivity of invasive GBS isolates. A total of 490 patients participated in this multicenter study; 160 had urinary tract infection, 115 complained of diabetic foot ulcers, 125 men had genital tract infections, and 30 women females had genital tract infections. Others had bronchopneumonia, otitis media, synovitis, or meningitis. Serotyping of the isolated GBS was performed at the CDC in the United States. Antibiotic sensitivity patterns were determined using the disk diffusion method. In men, the most common serotypes were II, III, and V, whereas types Ia, II, III, and V were isolated from women. Macrolides (erythromycin) resistance occurred in 4.1% of the isolates; 10.2% were resistant to both clindamycin and inducible resistance of macrolides, lincomycin, and streptogramin; 17.3% were resistant to quinolones; and 95.9% were resistant to tetracyclines. GBS primarily infected the urinary tract, skin, soft tissue, and genital tract in both genders. Isolates were sensitive to beta-lactam drugs, vancomycin, and linezolid; 14.0% were resistant to macrolides with or without clindamycin. Only 6.0% of the strains were sensitive to tetracyclines. Although GBS causes invasive infections in Egyptian adults, it rarely causes neonatal meningitis or sepsis. Future studies should determine whether GBS isolates are transmitted sexually, by performing a follow-up study of the partner of the infected patient.

INTRODUCTION

Streptococcus agalactiae [group B Streptococcus (GBS)] is within the phylum Firmicutes.¹ Optional, fastidious, beta-hemolytic cocci, GBS can be found in normal genitourinary, rectal, and oral flora.² Based on the cell wall capsular polysaccharide component, GBS is classified into 10 serotypes: Ia, Ib, II, III, IV, V, VI, VII, VIII, and IX.³

Epidemiological studies conducted in East African countries showed GBS colonization rates ranging from 3.0% to 28.8%; Central Africa, 20.0%; western Africa, 2.5% to 34.2%; southern Africa, 1.8% to 48.2%; and northern Africa, 17.0% to 26.5%.⁴ Furthermore, most patients with GBS invasive infection have at least one chronic debilitating disease—in particular, diabetes mellitus, which is probably a result of its associated leukocyte dysfunction.⁵

Invasive disease resulting from GBS infection has greatly increased in non-pregnant adults during the past decade and includes bacteremia, endocarditis, and infections of soft tissue, bones, and joints.² There are considerable differences among countries in the distribution of GBS serotypes. Recent research has focused on the molecular and epidemiological qualities of invasive GBS isolates.^6–8

To our knowledge, no published data exist on the incidence of invasive GBS disease and its antimicrobial susceptibility. This study aimed to determine the serotype and antibiotic susceptibility of GBS isolates from Egyptian patients to determine their serotype and antibiotic susceptibility.

MATERIALS AND METHODS

Study design.

Between September 2016 and December 2019, 490 patients age 24 to 53 years old were recruited for the prospective observational multicenter study. All patients were Egyptians and came from rural areas. A total of 160 patients had urinary tract infections with or without diabetes mellitus (120 women and 40 men); 115 patients complained of diabetic foot ulcers (skin and soft tissue infection; 60 women and 55 men); 125 men had genital tract infections in the form of prostatitis, urethritis, and pyospermia; and 30 women had genital tract infections manifested by increased white blood cells in their vaginal discharge. Forty patients had bronchopneumonia (10 women and 30 men), otitis media (10 men), synovitis (five men), or meningitis (five men) (Table 1, Figure 1).

Table 1.

Group B Streptococcus serotype distribution between females and males with multisystem affectation (N = 490)

GBS serotype	UTI		Diabetic foot		GTI		Bronchopneumonia		Otitis media		Synovitis		Meningitis		n
	Female (n = 120)	Male (n = 40)	Female (n = 60)	Male (n = 55)	Female (n = 30)	Male (n = 125)	Female (n = 10)	Male (n= 30)	Female (n = 0)	Male (n = 10)	Female (n = 0)	Male (n = 5)	Female (n = 0)	Male (n = 5)
Ia	40 (33.3%)	5 (12.5%)	15 (25.0%)	10 (18.2%)	5 (16.7%)	15 (12.0%)	–	5 (16.7%)	–	–	–	–	–	–	95
Ib	10 (8.3%)	–	–	5 (9.1%)	–	5 (4.0%)	–	–	–	–	–	–	–	–	20
II	20 (16.7%)	25 (62.5%)	10 (16.7%)	15 (27.3%)	5 (16.7%)	15 (12.0%)	5 (50.0%)	5 (16.7%)	–	–	–	–	–	–	100
III	30 (25.0%)	5 (12.5%)	5 (8.3%)	–	10 (33.3%)	50 (40.0%)		5 (16.7%)	–	–	–	–	–	–	105
IV	–	–	–	–	–	–	–	5 (16.7%)	–	–	–	–	–	–	5
V	10 (8.3%)	5 (12.5%)	25 (41.7%)	20 (36.3%)		35 (28.0%)	5 (50.0%)	5 (16.7%)	–	5 (50.0%)	–	–	–	–	110
VI	10 (8.3%)	–	–	5 (9.1%)	5 (16.7%)	5 (4.0%)	–	–	–	–	–	–	–	–	25
VII	–	–	5 (8.3%)	–	5 (16.7%)	–	–	–	–	5 (50.0%)	–	5 (100%)	–	5 (100%)	25
VIII	–	–	–	–	–	–	–	5 (16.7%)	–	–	–	–	–	–	5

GBS = group B Streptococcus; GTI = genital tract infection; UTI = urinary tract infection.

Figure 1.

Age and gender distribution of the enrolled patients. This figure appears in color at www.ajtmh.org.

Before the study was conducted, the purpose and objectives of the study were described to students and the study participants or guardians. Prior to any procedure in specialized treatment centers (including the Al-Hussein University Hospital in Cairo), all participants provided informed consent. Study practices were consistent with good clinical practice guidelines and the Declaration of Helsinki. The study also received approval from the National Liver Institute Institutional Review Board (IRB no. 00003545, University of Menoufia). All participant information was kept confidential.

Diabetes mellitus was diagnosed according to the WHO and the American Diabetes Association as fasting blood glucose ≥ 126 mg/dL or 2-hour post prandial blood glucose ≥ 200 mg/dL.⁹

Data regarding age, gender, residence, education, job position, past medical history, current symptoms, and other risk factors were recorded and collected through a written questionnaire adapted from the Exposure Prevention Information Network (http://www.healthsystem.virginia.edu/internet/epinet).

Exclusion criteria.

Patients treated with antibiotics 2 weeks before recruitment or with a complex past or present history of pregnancy (abortion, premature delivery, premature rupture of membranes) were excluded from the study.

Sample collection and bacterial identification.

Excessive secretions were removed from vaginal specimens, and mucus from the lowest third of the vagina was removed without a speculum, as recommended by the CDC (2010).⁶⁰ Clean-catch voided urine and genital samples were subjected to direct microscopic examination for the presence of white blood cells, and all samples were stained with Gram and Giemsa stains. Sterile swabs were taken from foot ulcers of diabetic patients, bronchoalveolar lavage fluid from patients with bronchopneumonia, ear swab and pipette-aspirated specimens from patients with otitis media, blood specimens from patients with endocarditis, and cerebrospinal fluid from patients with meningitis.

Specimens were cultivated on double-layer sheep blood agar plates to isolate GBS by the beta-hemolytic activity on red blood cells. GBS colonies appear gray or whitish gray and are surrounded by a weak zone of hemolysis in blood culture media. Bacterial isolates were also characterized using the coagulase and catalase tests. Latex agglutination tests confirmed the presence of GBS.

Serotyping.

Samples were sent to the CDC in Atlanta, Georgia, for serotyping of GBS isolates. Samples were transported using a single rabbit anti-bulb kit (Denka Seiken, Tokyo, Japan) according to the manufacturer’s protocol.

Antimicrobial susceptibility testing.

The disk diffusion method was used to assess antibiotic sensitivity patterns of the GBS isolates (Kirby and Bauer method).⁶¹ The types of antibiotics and their concentrations were used per Clinical and Laboratory Standard Institute guidelines:¹⁰ ampicillin; amoxicillin; penicillins; cefotaxime; linezolid; vancomycin; erythromycin; clindamycin; inducible macrolides, lincomycin, and streptogramin (iMLS); quinolones; and tetracyclines. Streptococcus pneumoniae ATCC 49619 was used as a quality control strain.

Statistical analysis.

SPSS (v. 22.0; IBM, Armonk, NY) was used for all data analyses. Descriptive statistics include mean, percentage, and SD.

RESULTS

GBS strains were isolated from all 490 patients [age range, 24–53 years; 220 women (44.9%) and 270 men (55.1%)].

Isolation of invasive GBS.

GBS strains were isolated from all 490 patients and identified by colony characteristics, type of hemolysis, Gram stain, and catalase test results. The final diagnosis was confirmed by the latex agglutination test (Oxoid). GBS caused urinary tract, soft tissue, and genital tract infections in more than 80% of the male and female patients in our study. Women with diabetes were more prone to GBS urinary tract infection, especially those in their childbearing years as opposed to their menopausal years.

Serotyping of GBS.

All nine GBS serotypes were isolated from all samples from male and female patients. Serotypes Ia, II, III, and V were isolated most isolated, representing 19.4%, 20.4%, 21.5%, and 22.4%, respectively, of all isolates. In the male patients, the most common GBS serotypes, in descending order, were V, II, III, and Ia; in females, the most common isolates were Ia, III, II, and V (Table 1).

There was a marked difference between the GBS serotypes isolated from male and female patients with urinary tract infections. In descending order, Ia, III, II, Ib, V, and VI were isolated from female patients, whereas types II, Ia, III, and V were isolated from male patients (Table 1). GBS serotypes isolated from male genital tract infections were III, V, Ia, II, Ib, and VI, in descending order. In female patients, the GBS serotypes were III, Ia, II, VI, and VII, in descending order (Table 1). Serotypes Ia, II, and III were isolated from female urinary tract (75%) and genital tract (67%) infections of other patients (Tables 1). GBS serotypes were more or less similar in diabetic and non-diabetic female patients with urinary tract infections (Table 2). A marked difference was detected between the serotypes isolated from patients with prostatitis (serotypes III, V, II, Ia, and Ib), urethritis (III, V, Ia, and II), and pyospermia (Ia, III, V, and VI) (Table 3).

Table 2.

Group B Streptococcus types of urinary tract infection in female patients with or without diabetes (N = 120)

GBS serotype	Group
	Diabetes (n = 50)		No diabetes (n = 70)
	n	%	n	%
Ia	10	20	30	42.9
Ib	5	10	5	7.1
II	10	20	10	14.3
III	10	20	20	28.6
V	5	10	5	7.1
VI	10	20	0	0

GBS = group B Streptococcus.

Table 3.

Group B Streptococcus types isolated from male genital tract infection (N = 125)

GBS serotype	Group
	Prostate (n = 70)		Urethral discharge (n = 35)		Semen (n = 20)
	n	%	n	%	n	%
Ia	5	7.1	5	14.3	5	25.0
Ib	5	7.1	0	0.0	0	0.0
II	10	14.3	5	14.3	0	0.0
III	30	42.9	15	42.9	5	25.0
V	20	28.6	10	28.6	5	25.0
VI	0	0.0	0	0.0	5	25.0

GBS = group B Streptococcus.

GBS serotypes isolated from pus collected from diabetic feet in female and male patients were, in descending order, V, Ia, II, III, and VII in women, and serotypes V, II, Ia, Ib, and VI in men (Table 1). GBS serotypes II and V were isolated from bronchopneumonia samples of both genders, whereas types Ia, III, IV, and VIII were isolated from men only (Table 1). In 10 male patients with otitis media, serotypes V and VII were isolated; serotype VII was isolated from five male patients with synovitis and five men with meningitis (Table 1).

Antibiotic susceptibility test for the isolated GBS.

All GBS serotypes isolated were sensitive to beta-lactam drugs (ampicillin, amoxicillin, penicillins, and cephalosporins), linezolid, and vancomycin. Although 4.08% were resistant to the macrolides (erythromycin) and 10.2% were resistant to clindamycin and iMLS, 17.3% were resistant to quinolones and 95.9% were resistant to tetracyclines.

DISCUSSION

GBS is one of the primary causes of neonatal sepsis and meningitis, and is a potentially invasive infectious agent of pregnant and non-pregnant adults.^11,12 The serological reaction against the polysaccharide capsule of GBS9 or multiplex polymerase chain reaction results can identify 10 serotypes (Ia, Ib, and II–IX) into 10 different serotypes.¹³

The serotype isolated most commonly from patients enrolled in our study was type V, followed by types III, II, and Ia, representing 22.4%, 21.5%, 20.4%, and 19.4%, respectively, of all samples. These results are consistent with another study conducted in Egypt by Shabayek et al.¹⁴ In Kuwait, Boswihi et al.¹⁵ reported that serotype V was isolated from 38.5% of 143 GBS isolates, followed in frequency by serotypes III (20.9%), Ia (7.7%), and II (11.2%). Serotypes Ib and IV each accounted for only 3.5%, and VI and VII constituted 2.8% and 0.6%, respectively.¹⁵

In Europe and North America, serotype III is a common colonizer in Canada compared with our study.^16–20 In Malaysia, serotype IV strains predominated and were the second most common isolate in adults with skin infections.^21,22 In Gabon, serotype V was the most common, and in the Republic of The Gambia, serotype VI was the most common.^23,24 The most common serotypes in Taiwan were III and VI; and in Japan, VI and VIII.^25–27 Serotype IX was recently reported from Denmark.²⁵ The distribution and prevalence of certain serotypes can change over time.

Four serotypes (Ia, II, III, and V) represented 83.7% of all isolates in our study. Similarly, Ji et al.²⁸ found that six serotype III serotypes represented 54.9% of 153 GBS isolates. The contribution (Ia, Ib, III) of total serotypes, representing 85.6%, is greater than that found in a study conducted in Beijing (75.1% of all serotypes). Lu et al.^29–35 found that serotype II (7.0% of total serotypes) was also greater (69.2%).

The distribution and resistance of GBS serotypes shifted between serotype V and serotype Ia as the dominant GBS serotype of infected women. Although the most prevalent serotype found in our study, Ia, is of increasing concern in Egypt, serotype V GBS isolates most often led to invasive inflammation in patients, adults, and neonates in the United States and Canada,^36,37 and Australia and New Zealand.³⁸

In the male patients, the most common SGB type was V, followed by II, III, Ia, and VII, whereas type Ia was isolated most commonly from the female patients (27.3%). Type V was isolated from only 18.2% of women. Suara et al.³⁹ (1994) found that of 32 infected pregnant women, 12 (30%) were colonized with serotype V. Serotype V has been identified as globally important in neonatal and adult colonization disease and serotype disease.^31,40,41

In our study, 125 of 160 isolates (78.1%) from patients with urinary tract infections were GBS serotypes Ia, II, and III.^14,42,43 These results are consistent with one of two previous studies conducted in Egypt (and were higher than those reported in the second study).^44,45 The most prevalent serotypes in the study by Ulett et al.⁴⁶ were in the 62 cases of GBS urinary tract infections, with serotypes Ia, III, and V representing 76% of the cases. The remaining 24% of the cases were isolates of serotypes Ib, II, and IV.

Global GBS serotype variability is linked to different sociocultural, geographic, climatic, biological, and methodological factors, emphasizing the importance of individualization, local infection rates, and preventive strategies.

GBS-attributed skin and soft tissue infections can manifest as cellulitis, abscesses, foot infections, or ulcers. Diabetes mellitus is a common underlying disease in patients infected with GBS and with soft tissue infections.⁴⁷

Severe invasive disease has been linked to serotypes Ia, Ib, II, III, and, most recently, V.¹ In our study, GBS serotypes isolated from pus collected from diabetic feet in both female and male patients were, in decreasing prevalence, serotypes V, Ia, II, III, and VII, and serotypes V, II, Ia, Ib, and VI, respectively. Consistent with our study, Lee et al.³⁵ reported types V, II, and Ia to be the most common GBS serotypes causing soft tissue infections; 82.6% of these serotypes caused diabetes foot in both genders.

The most common isolated serotype isolated from genital infections was III in both genders, representing 38.7% (60 of 155 isolates), followed by isotype V with 22.6% (35 of 155 isolates). Serotype III predominated (50.9%), followed by serotype II, in a study of Malaysian 19 adult and neonate patients with genitourinary infections. Serotype III is related to a greater risk of newborn infection with more persistent colonization.⁴⁸

GBS serotypes II and V were isolated from bronchopneumonia specimens from both genders, whereas types Ia, III, IV, and VIII were isolated from the men only. Foster-Nyarko et al.⁴⁹ had similar results when evaluating the infection rates, serotypes, and antibiotic GBS susceptibility in 1,200 2-month-old children. They found that serotypes V and II were the dominant GBS serotypes. Nine GBS serotypes, most frequently V (37.6%) and II (34.6%), composed approximately 72% of all identified GBS serotypes. The other GBS serotypes were Ia (7.5%), IV (6.0%), Ib (5.3%), VII (3.8%), VIII (0.8%), III (0.8%), and VI (0.8%). Deng and Yang^50–52 found serotypes Ia, III, and II were the most prevalent, particularly in cases of identified neonatal fatal pneumonia.

All tested isolates were sensitive to beta-lactam drugs (ampicillin, amoxicillin, penicillins, and cephalosporins), linezolid, and vancomycin. Although 4.08% were resistant to the macrolides (erythromycin) and 10.2% were resistant to clindamycin and iMLS, 17.3% were resistant to quinolones and 95.9% were resistant to tetracyclines. A study in Nigeria reported an extremely high level of penicillin and ampicillin resistance (100%).^53–55 The authors considered the high resistance rate the consequence of the ease by which antibiotics are procured in developing countries. They concluded that there was widespread therapeutic antibiotic use, and this practice may be justified to a certain extent. Some women with penicillin allergies require other GBS prophylactic antibiotics, such as clindamycin, erythromycin, or vancomycin.

Alternative antibiotics, including clindamycin, erythromycin, or vancomycin, are administered to pregnant women with penicillin allergies. Multiple studies have shown the resistance to clindamycin and erythromycin is increasing.^{14,42,51–54}

Shabayek et al.⁴⁰ found that 13.15% of GBS isolates were erythromycin resistant and 23.7% were clindamycin resistant. In the study of Shabayek et al., erythromycin, and clindamycin resistance rates were much lower than those reported in Syria by Dunia et al.⁵⁶ However, these antibiotics are at higher rates than those reported for erythromycin.⁵⁷

No isolates studied by Sadaka et al.⁴³ were resistant to penicillins such as penicillin and ampicillin. Moreover, all isolates have ceftriaxone, cefepime, vancomycin, linezolid, and cefotaxime. Sadaka et al.⁴³ found the resistance to levofloxacin, azithromycin, erythromycin, and clindamycin was 43.4%, 28.3%, 22.6%, and 15%, respectively. Eleven GBS isolates (20.8%) were intermediately tetracycline susceptible.⁴³

In a recent Egyptian study conducted by Foad,⁵⁸ (2016), GBS was very vulnerable to ampicillin, benzylpenicillin, cefotaxime, ceftriaxone, linezolid, trimethoprim/sulfamethoxazole, and vancomycin. In isolates from urinary tract and vaginal infections, levofloxacin demonstrated good susceptibility (82.1% and 78.9%, respectively). Erythromycin and clindamycin, however, demonstrated a moderate urinary and vaginal resistance (53.6% and 68.4%, respectively) and (57.1% and 63.2%, respectively) to clindamycin. The rate of resistance in urinary and vaginal infections was very high (96.4% and 94.7%, respectively).⁵⁸

Penicillin and ampicillin, both first-line treatments for pregnant women and neonates, have been found to be efficient in the treatment and prevention of GBS.⁵⁹ Therefore, penicillin is less likely to select resistant organisms and has a narrow range of antimicrobial activity. Penicillin, ceftriaxone, or vancomycin were susceptible to 100% of isolates. Cefazoline, clindamycin, and erythromycin may be useful alternatives for women allergic to penicillin. Ji et al.²⁸ found 64.9% were resistant to erythromycin, 52.4% to clindamycin, and 25.9% to levofloxacin. Clindamycin and levofloxacin resistance has increased significantly during the 6 years of monitoring. However, in the past 20 years, the proportions of GBS isolates resistant to erythromycin, clindamycin, or levofloxacin have increased considerably.⁶⁰ Most previous studies show GBS isolates had a high rate of resistance to tetracycline.

CONCLUSION

Although GBS causes invasive infections in Egyptian adults, it rarely causes neonatal meningitis or sepsis. GBS infects mainly the urinary tract and skin and soft tissue in both genders. However, the genital tract was more infected in men than in women in our study. Diabetic young females (of childbearing age) are more likely to be infected with GBS than older women.

Many GBS isolates were found to be sensitive to beta-lactam drugs, vancomycin, and linezolid, whereas only 14% were resistant to macrolides with or without clindamycin, 17.3% were resistant to quinolone, and only 4% of the strains were sensitive to tetracyclines.

Future studies should determine whether GBS isolates are transmitted sexually, by performing a follow-up study of the partner of the infected patient. Furthermore, it is essential to assess whether GBS may cause autoinfection in patients who may have a GBS focus elsewhere in their body. The most significant limitation of our study was that pregnant women could not follow the positive GBS culture to determine premature diabetes and infant infection rates.