Although many studies reported the importance of GBS in human and veterinary medicine transmission between human and animal or vice versa is poorly understood24. Furthermore, molecular epidemiological studies that compare the phenotype and genotype properties of GBS involving different origins, particularly human and fish origin, are scarce in South East Asia.
The multidrug-resistant isolates have been increasing due to the intensive culture of fish and antimicrobial misuse to control bacterial infection in South East Asia. According to the Centers for Disease Control and Prevention (CDC) in the US, GBS isolates are completely susceptible to beta-lactam antibiotics include penicillin derivatives and cephalosporins. At the same time, CLSI reports that non-susceptible GBS isolates to beta-lactams are sporadic25. Despite this, penicillin resistance in GBS has been reported from Asia, including in human isolates in Japan and from tilapia isolates in China26,27. GBS antimicrobial resistance profiles differed among humans and fish origin.
In this study, we reported resistance to erythromycin, azithromycin, clindamycin, chloramphenicol and ofloxacin among human GBS isolates, while all fish GBS isolates were susceptible to these antibiotics. This contrasts with results reported from China, where erythromycin resistance was higher among GBS isolated from cultured fish28,29. In contrast, the fish isolates were higher in resistance to tetracycline in this study which may be because tetracycline is widely used to treat several bacterial infections in freshwater fish30. These results were similar to previous studies in Southeast Asia among human and fish origin31,32.
In addition, the antimicrobials penicillin, amoxicillin, vancomycin, ceftriaxone, cefotaxime, cefepime, and linezolid showed largely efficacy against humans and fish GBS isolates, which were reported in previous studies as well11,18,33−35. Penicillin is the first-line antibiotic for treating and prophylaxis GBS infections among humans; however, erythromycin and clindamycin are the alternative antibiotics for allergic patients allergic to penicillin35. In our study, the resistance rates to erythromycin and clindamycin among human isolates were lower than those reported in other studies in major hospitals in Malaysia10,11. An increase of resistance to erythromycin, clindamycin and azithromycin are commonly used in humans may be needed to concern. GBS human isolates had a common multidrug resistance pattern of resistance to these antibiotics, which was also identified in other studies10,11,35.
In this study, cps gene serotyping revealed that there was a difference in serotype distribution among GBS human and fish isolates in this study. There was a different variety of serotypes among the human isolates. The most common serotype was serotype V, followed by Ia, II, VI and III. Furthermore, serotype VIII did not occur among the human isolates, while serotype Ix was the lowest. Previous studies showed serotypes V and Ia as the common serotypes among GBS human invasive isolates from sterile isolation sites 9,11,36. However, serotype III was the most common (87.15%), and it was detected mainly in septicemia cases among invasive GBS in Thailand. In another study in Malaysia, the most prevalent serotype from sterile and non-sterile isolation sites VI, followed by VII, III, Ia and V10. Generally, serotype Ia and V are commonly present in non-pregnant adults37. In addition, serotypes III and V are usually associated with invasive GBS infections38. On the other hand, only serotype III was identified from all 114 fish GBS isolates. Other studies reported serotype III was dominant in tilapia in Malaysia, Thailand, Vietnam and Brazil7,39−42. However, a study in Brazil reported that was serotype Ib was the dominant34, while another study in China found serotype Ia to be the dominant44. Moreover, a similar was found between serotype III isolated from red tilapia with the GBS found in the Singaporean outbreak, possibly due to cross-host pathogenesis of the strains. Globally, there are marked regional differences in the distribution of GBS serotypes.
The virulence and pili genes promote GBS to attack, destroy and invade the host cells and evade the immune system. The present study showed that cfp, cylE, hylB, fbsA, fbsB and spb1 genes were present in nearly 100% of the human and fish isolates. We observed a difference between GBS human and fish isolates in the possession of virulence genes Imb, scpB, rib, bac, PI-1, PI-2a and PI-2b. The scpB, lmb and rib genes are usually found in human GBS isolates and absent in fish and bovine isolates45. PI-2a was completely missing from the fish isolates, similarly to previous46. This difference is probably due to deletion during putative adaptation of GBS to the fish host45,47. Thus, it might be possible to characterize the strains of different host origins using the virulence genes profiles. According to the virulence genes profiling of GBS, human isolates are believed to possess a higher capability to adhere and invade the host cells compared to fish isolates. However, the immune evasion functions of fish isolates could be higher than human isolates.
This study revealed 19 different STs among human and fish isolates, including three newly assigned STs. ST1 was the most prevalent among the human isolates and belonged to CC1, while only ST283 was found in all fish isolates. This is in line with a study in Malaysia that reported ST1 was the most common ST associated with invasive human isolates13. Another study in Taiwan also reported that ST1 (26.0%) was the most prevalent STs as a leading cause of invasive disease that affects non-pregnant adults43. ST283 were the common STs related to epidemiological studies and disease outbreaks in fish42,44,48. In Malaysia, a study of MLST assays showed that eight and 50 isolates of GBS were represented by ST7 and ST283 only, respectively7. MLST data of GBS from fish in Malaysia is still lacking, as well as for human isolates. According to a study conducted in China, GBS ST7 has been identified as a highly virulent strain that causes meningoencephalitis in tilapia49. In addition, Thailand has reported the case of GBS III ST283 infection in tilapia, which causes significant economic losses to their aquaculture industry6. In the current study, GBS III ST283 was found in two cases among the human isolates, which were associated with Chinese ethnicity, non-pregnant patients and susceptibility to antibiotics. Similar studies reported that GBS ST283 was more associated with eating raw fish, Chinese ethnicity and non-pregnant patients20,21. To our knowledge, we report GBS III ST283 in humans for the first time in Malaysia. Meanwhile, GBS ST283 has also been isolated from human clinical samples in Thailand has demonstrated which were closely related to the Singaporean outbreak strain in 2015, and it has been demonstrated that ST283 is a zoonotic GBS clone that causes severe invasive disease in humans21. CC283 was associated with the type B gbs2018 gene emergence as a human pathogen50. This highlights the threat of continued evolution and expansion of the host range of GBS. Furthermore, CC1 was identified as a common cause of GBS septicemia induced by consuming contaminated seafood in France51. Based on the examples presented here, it is evident that there are no true species barriers, at least at the ST level. The risk of transmission of GBS between fish and people can be reduced by improving hygiene and cooking food properly.
The phylogenetic tree indicated a genetic linkage between two human and fish isolates sharing a similar lineage of CC283 in clade I. All isolates in CC283 showed non-multidrug resistance pattern and serotype III as well, suggesting that the strains isolated from the human was likely to originate from fish. In bacteria, diversity is associated with mutations, transformation, transduction, or conjugation involving horizontal DNA transfer. GBS genetic diversity was primarily due to recombination, which can lead to altered serotypes, virulence, and host range52,53. Our results also placed CC12 and CC3 near CC283 on the phylogenic tree. The most dominant clade I consisted of isolates that exhibited ST283, ST12, ST130, ST1670, ST485, and ST3 with different serotypes; serotype III was the most prevalent in clade I. Clade II consisting of human GBS alone isolates, was the second major clade representing ST28, ST861, ST335, ST19, ST1668, ST196, ST459, ST1 and ST1669. There was a difference in serotype distribution and MDR phenotype in this clade. Meanwhile, clade III consisted of three ST26 isolates with similar serotype V, non-MDR, and they were both from blood, which reflected their close association. At the same time, clade IV represented ST24, ST23 and ST17. The most common serotype in this clade was III, followed by Ia. All the isolates in this clade were non-MDR. The new ST1670 belonged to the CC12 lineage, while ST1668 and ST1669 belonged to CC1 in the phylogenetic tree, having serotype Ia, VI and II, respectively, and non-MDR. CC17 could emerge differently from others as it was located more basally and distinct in the tree structure. In this study, phylogenetic tree is consistent with previous study54, in which GBS isolated from different hosts exhibited high genetic diversity, with few relationships between genotypes. All in all, of the human isolates, there are only two GBS ST 283. Therefore, these strains should be monitored sequentially throughout Malaysia, and the additional fish-origin isolates need to be characterized. Further analysis utilizing whole-genome sequencing is highly warranted to elucidate this matter and provide a better understanding of the genetic organization and evolution of the human and fish isolates.