Agalactiae Isolates Research Articles

Host range, morphological and genomic characterisation of bacteriophages with activity against clinical Streptococcus agalactiae isolates.

Streptococcus agalactiae or Group B Streptococcus (GBS) is a leading cause of sepsis in neonates. As a preventative measure prophylactic antibiotic administration is common in pregnant women colonised with GBS, but antibiotic-resistance and adverse effects on neonatal microbiomes may result. Use of bacteriophages (phages) is one option for targeted therapy. To this end, four phages (LF1 –LF4) were isolated from wastewater. They displayed lytic activity in vitro against S. agalactiae isolates collected from pregnant women and neonates, with 190/246 isolates (77.2%) and 10/10 (100%) isolates susceptible to at least one phage, respectively. Phage genomes ranged from 32,205–44,768 bp and all phages were members of the Siphoviridae family. High nucleotide identity (99.9%) was observed between LF1 and LF4, which were closely related to a putative prophage of S. agalactiae. The genome organisation of LF2 differed, and it showed similarity to a different S. agalactiae prophage, while LF3 was more closely related to a Streptococcus pyogenes phage. Lysogenic gene presence (integrase, repressor and regulatory modules), was suggestive of temperate phages. In a therapeutic context, temperate phages are not ideal candidates, however, the broad host range activity of these phages observed on clinical isolates in vitro is promising for future therapeutic approaches including bioengineered phage or lysin applications.

Antibiotic Susceptibility Patterns and Prevalence of Streptococcus Agalactiae Rectovaginal Colonization Among Pregnant Women in Iran.

Streptococcus agalactiae is an important pathogen in neonates and pregnant women. Neonatal invasive infections due to S. agalactiae are life-threatening and preventive strategies for this challenge of human have become a concern. The aim of the present study was to determine the prevalence of rectovaginal colonization, related risk factors and antibiotic resistance pattern of S. agalactiae among pregnant women in Iran. The present study was performed on 240 pregnant women. Vaginal and rectal swabs were obtained from all of the women and then were transferred to the laboratory. The isolation and identification of S. agalactiae was performed by standard microbiological tests and polymerase chain reaction (PCR) assay. The antimicrobial susceptibility patterns of the isolates were determined by the Kirby-Bauer disk diffusion. Polymerase chain reaction was used to detect ermB and mefA genes in erythromycin-nonsusceptible isolates. Out of 240 pregnant women, 16 cases (6.7%) were colonized by S. agalactiae. There is no significant association between demographic-obstetric factors and maternal S. agalactiae colonization in the pregnant women. Linezolid, vancomycin and ampicillin were the most effective antibiotics against S. agalactiae. The ermB gene was present in 6 (35.29%) S. agalactiae isolates. However, the mefA gene was not detected in any of the isolates. Given the relatively significant prevalence of S. agalactiae colonization in the pregnant women in the present study and the risk of serious neonatal infections, the screening of pregnant mothers for the bacteria seems necessary. Our findings highlight the importance of appropriate antibiotic prophylaxis during pregnancy for the prevention of early onset S. agalactiae-neonatal infection and comorbidity.

Gangrenous mastitis in dromedary camels in UAE caused by Streptococcus agalactiae

BackgroundMastitis is a disease of economic concern that affects dairy industry worldwide. This study aimed to investigate and identify possible etiologies encountered in an episode of acute gangrenous mastitis in lactating she-camels in Al Dhafra region, Abu Dhabi Emirate, United Arab Emirates (UAE).Beside the routine clinical examination, conventional bacteriological methods were used to isolate and identify possible aerobic/anaerobic bacterial or fungal pathogens from cultured milk samples collected from the mastitic she-camels. Moreover, quantitative real-time polymerase chain reaction (qPCR) was used for the detection of Mycoplasma agalactiae and Mycoplasma bovis strains, and the 16S rRNA gene was sequenced to confirm the isolation. The isolates were also tested for their susceptibility to antimicrobials.ResultsAcute gangrenous mastitis is reported in the dromedary camel herd with about 80% morbidity rate among lactating she-camels exhibited acute, painful hard swelling of affected teat, quarter or entire udder. About 41.7% of the infected animals were stamped out for culling due to complete or partial amputation of udder quarters. Streptococcus agalactiae was the sole isolated organism (6 isolates). The antimicrobial susceptibility testing revealed that, the Streptococcus agalactiae isolates were sensitive to both penicillin and ampicillin. Comparison of the 16S rRNA gene sequencing results by BLASTN confirmed the presence of Streptococcus agalactiae with high confidence (100% identity). Phylogenetic analysis indicated clustering of one isolate (CMAUAE accession number; MN267805.1) with Streptococcus agalactiae that infects multi-hosts including humans, while strains (CMBUAE to CMFUAE with accession numbers; MN267806.1 to MN267810.1 respectively) clustered with Streptococcus agalactiae that infects humans. No Mycoplasma spp was detected by qPCR analysis.ConclusionsIn the present study, the Streptococcus agalactiae was found to be the main cause of acute gangrenous mastitis in dromedary camels in UAE. More research should be done to investigate other possible causes of clinical or subclinical mastitis in dromedary camels in UAE.

Characterization of Staphylococci and Streptococci Isolated from Milk of Bovides with Mastitis in Egypt.

The aim of this study was to characterize staphylococci and streptococci in milk from Egyptian bovides. In total, 50 milk samples were collected from localities in the Nile Delta region of Egypt. Isolates were cultivated, identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and antibiotic susceptibility testing was performed by the broth microdilution method. PCR amplifications were carried out, targeting resistance-associated genes. Thirty-eight Staphylococcus isolates and six Streptococcus isolates could be cultivated. Staphylococcus aureus isolates revealed a high resistance rate to penicillin, ampicillin, clindamycin, and erythromycin. The mecA gene defining methicillin-resistant Staphylococcus aureus, erm(C) and aac-aphD genes was found in 87.5% of each. Coagulase-negative staphylococci showed a high prevalence of mecA, blaZ and tetK genes. Other resistance-associated genes were found. All Streptococcus dysgalactiae isolates carried blaZ, erm(A), erm(B), erm(C) and lnuA genes, while Streptococcus suis harbored erm(C), aphA-3, tetL and tetM genes, additionally. In Streptococcus gallolyticus, most of these genes were found. The Streptococcus agalactiae isolate harbored blaZ, erm(B), erm(C), lnuA, tetK, tetL and tetM genes. Streptococcus agalactiae isolate was analyzed by DNA microarray analysis. It was determined as sequence type 14, belonging to clonal complex 19 and represented capsule type VI. Pilus and cell wall protein genes, pavA, cadD and emrB/qacA genes were identified by microarray analysis.

Subclinical mastitis in pastoralist dairy camel herds in Isiolo, Kenya: Prevalence, risk factors, and antimicrobial susceptibility

Mastitis is an important constraint to milk production in pastoralist camel (Camelus dromedarius) herds in Kenya. The objective of this study was to investigate the prevalence, risk factors, and bacterial panorama of subclinical mastitis (SCM) in pastoralist camel herds in Isiolo County, Kenya. Furthermore, antimicrobial susceptibility in udder pathogens was studied. A cross-sectional sample of 206 camels from 20 milking herds was screened using the California Mastitis Test (CMT), and quarter milk was subjected to bacterial culturing. Isolates were confirmed using MALDI-TOF mass spectrometry analysis, and antimicrobial susceptibility was determined using the broth microdilution method. Interviews focusing on herd management were conducted with camel owners. Subclinical mastitis, defined as a CMT score ≥ 3 (scale 1 to 5) and absence of clinical symptoms in the udder, were present in all visited herds. On the individual level, 46% of the camels had at least 1 quarter affected with SCM, and on the quarter level the prevalence was 26%. Intramammary infections (IMI) were common; out of 798 quarter milk samples, 33% yielded conclusive bacterial growth. The sensitivity and specificity of CMT for correctly identifying quarters with IMI were 82% and 92%, respectively. The most prevalent pathogen was Streptococcus agalactiae (72% of IMI-positive quarters), followed by non-aureus staphylococci (19%) and Staphylococcus aureus (13%). Antimicrobial susceptibility testing revealed that only a low proportion (4.9%) of Strep. agalactiae isolates was sensitive to tetracycline. For Staph. aureus, 59.1% of isolates exhibited sensitivity to penicillin. Skin lesions on the teats or udder were a risk factor for SCM. Increased age, parity, and stage of lactation were associated with increased risk of both SCM and IMI. Older camels with a blind teat or a previous history of mastitis were more likely to be infected with Strep. agalactiae. Hygiene routines for milking were largely absent in the observed herds, and knowledge of adequate milk handling was limited. The poor udder health is likely to depend on multiple factors, most prominently the within-herd maintenance of contagious udder pathogens, in combination with difficult sanitary conditions and lack of awareness among camel keepers. This study showed that in pastoralist camel herds around Isiolo town, SCM and IMI specifically caused by Strep. agalactiae are common udder health problems and are associated with increasing age, parity, and stage of lactation, and skin lesions on the teats and udder. Resistance to tetracycline in Strep. agalactiae was common. Control strategies specifically targeting SCM and adapted to pastorally managed camel herds need to be developed to reduce disease, combat antimicrobial resistance, and improve the livelihoods of pastoralists.

Streptococcus agalactiae infections and clinical relevance in adults, Thailand

Streptococcus agalactiae is an important pathogen that causes infections in neonates and adults; infections especially in nonpregnant adults are increasing worldwide. Of 1736 S. agalactiae isolates from individuals throughout Thailand, serotypes III (46.4%) and V (21%) were demonstrated to be the most common serotypes. Human cases (56.5% female and 43.5% male) could be found all year round, with the peak occurring more frequently during the rainy season (May–October). The mortality rate of S. agalactiae infections was 11.6%, and serotype III was the most common serotype involved. Serotype III was strongly significantly (P value <0.001) correlated with meningitis (odds ratio [OR] = 26.72), sepsis (OR = 5.56), and septic arthritis (OR = 22.79). Serotype V was more associated with urinary tract infection than other serotypes. (P value = 0.005; OR = 2.32).

Iron Oxide Nanoparticles Reduced Biofilm Formation and Detection of lmb Genes in Streptococcus agalactiae Isolated From Patients with Diabetes Mellitus

Fifty eight isolates of Streptococcus agalactiae were obtained from wound and urine specimens among one hundred thirty six from patients with diabetes mellitus and identified through cultural, morphological, and biochemical examinations in addition to VITEK 2 Compact System. All of the isolates were tested regarding their sensitivity to 18 antibiotics and the results showed that all of the isolates were resist to clindamycin, and sensitive to teicoplanin. The current study was carried out to use two methods (i.e. microtiter biofilm and Congo red agar ) so as to provide and extend insight into bacterial colonization and biofilm formation among diabetic patients. The two methods were compared and the results revealed that microtiter was the best method for biofilm detection, and two chemical materials (i.e. silver oxide, and iron oxide) were used as anti-biofilms for all isolates, one of the best concentration and kind of chemical materials was Iron oxide at 75 mg/ml for reducing biofilm formation. PCR system was employed in order to detect the virulence genes (i.e lmb gene), the results that 30(51.72%) were positive for lmb genes.

Development and evaluation of a quantitative polymerase chain reaction for aquatic Streptococcus agalactiae based on the groEL gene.

The aim of this study was to develop a TaqMan quantitative polymerase chain reaction (qPCR), based on the Streptococcus agalactiae groEL gene, to specifically quantify levels of bacteria within samples derived from aquatic sources, particularly aquaculture. Enumeration of bacteria by qPCR was compared with culture-based methods. The qPCR was sensitive to 33 isolates of S. agalactiae, representing 11 clonal complexes from aquatic, bovine and human hosts. The specificity of the assay was 92·5% at a threshold Cq value of 35. No cross-reaction with Streptococcus iniae was noted and of the 22 comparator species screened to test assay specificity, Streptococcus porcinus had a Cq value of 33·7 S, while Streptococcus gallolyticus subsp. macedonicus and Streptococcus ictaluri had one replicate value above the Cq threshold of 35 (34·5 and 34·4 respectively), while only S. agalactiae were detected with a Cq value of 30. The limit of detection of the assay was 1·7 copies per µl at Cq 35. Discrepancies between molecular and culture-based methods of enumeration were noted. The qPCR was able to detect a diverse range of S. agalactiae isolates from different clonal complexes (CCs) and could distinguish between S. agalactiae and closely related species, notably S. iniae. The results suggest that a Cq 30 would be a very meaningful cut-off, allowing the detection of infected fish while ruling out all false positives. This rapid and sensitive qPCR assay is useful to quantify DNA copy number in the laboratory and could prove useful for detecting low levels of S. agalactiae in aquaculture systems, including Oreochromis niloticus culture.

Repetitive cellulitis caused by Streptococcus agalactiae isolates with different genotypic and phenotypic features in a patient having upper extremity with lymphedema after mastectomy and axillary lymph node dissection

Previously reported cases of recurrent cellulitis/erysipelas affecting chronically lymphedematous skin regions have been demonstrated to be due to Streptococcus agalactiae isolates with closely related genetic background which may be suggestive of relapse rather than reinfection. Herein, we report the occurrence of three episodes of repetitive cellulitis caused by S. agalactiae strains with different genotypic and phenotypic characteristics, including different antimicrobial susceptibility patterns (tetracycline, macrolide/lincosamide, and fluoroquinolone classes), in the left upper extremity of a patient with lymphedema, following left mastectomy and axillary lymph node dissection. The genotypic and phenotypic characteristics of the three isolates were confirmed based on the random amplified polymorphic DNA patterns, DNA profiles of virulence factors (bca-rib-bac-lmb-cylE), data on biofilm formation and cell invasion, antimicrobial susceptibility testing results, antimicrobial resistance (AMR) genotypes, and amino acid mutations associated with AMR. These results revealed that reinfection with S. agalactiae, rather than recurrence, occurred during the three episodes. In conclusion, microbiologic studies such as blood cultures or tissue cultures are certainly helpful in the management of recurrent infections or invasive infections such as bacteremia in order to better target antimicrobial therapy, regardless of the data previously presented.

In vitro evaluation of the antibacterial activities of radezolid and linezolid for Streptococcus agalactiae

PurposeThis study aims to evaluate the antimicrobial activities of linezolid and radezolid against Streptococcus agalactiae in vitro and compared for genetic resistance factors. MethodNonduplicate S. agalactiae clinical isolates (n = 136) were collected and the minimal inhibitory concentrations of antimicrobials were determined by agar dilution methodology. The linezolid-resistant mechanism in the clinical linezolid-non-susceptible S. agalactiae isolates and that induced by linezolid pressure in vitro were analyzed by PCR and sequence alignment. Antimicrobial activities and resistance mechanism distinctions between linezolid and radezolid were further investigated in the clinical linezolid-non-susceptible S. agalactiae isolates and that induced by linezolid pressure in vitro. ResultsOur data indicated that 17 (13%) of the 136 clinical S. agalactiae isolates were not susceptible to linezolid. For individual S. agalactiae isolates, including linezolid-nonsusceptible isolates with 23S rRNA V domain mutations, radezolid MIC90 values were generally one-half to one-quarter of the linezolid MIC90 values. Radezolid MICs remained low relative to linezolid MICs among linezolid-resistant S. agalactiae isolates, but exhibited the synchronous increases with the increasing copy numbers of 23S rRNA V domain mutations. Overall, 13 optrA-carrying clinical S. agalactiae isolates were found in this study and their MICs all remained sensitive to both linezolid and radezolid. Clinical S. agalactiae isolates with high radezolid MICs showed clonality clustering to sequence type (ST)10. ConclusionRadezolid exhibits stronger potency against S. agalactiae than linezolid and there is a concerning presence of linezolid-nonsusceptible S. agalactiae in clinical samples.

Genomic characterisation of perinatal Western Australian Streptococcus agalactiae isolates.

As a leading cause of neonatal sepsis, Streptococcus agalactiae, commonly known as Group B Streptococcus, is a major neonatal pathogen. Current global screening practices employ risk- or culture-based protocols for detection of these organisms. In Western Australia (WA), universal culture-based screening is provided, with subsequent intrapartum antibiotic prophylaxis for all S. agalactiae-positive women during labour. Widespread antibiotic exposure is not ideal and this is one of the factors driving development of vaccines against S. agalactiae. Vaccine candidates have focused on the capsule, surface proteins and pilus types, however, capsule serotypes are known to vary geographically. The aim of this study was to use genome sequencing to gain an understanding of the circulating genotypes in WA, and to assess variations in the associated gene pools. We sequenced 141 antenatal carriage (vaginal/rectal) isolates and 10 neonatal invasive disease isolates from WA. Based on the global PubMLST database, the 151 strains were characterised into 30 sequence types, with clustering of these mainly into clonal complexes 1, 12, 17, 19 and 23. Of the genes encoding eleven surface proteins that were analysed, the most prevalent were fbp, lmb and scpB which were present in ≥ 98% of isolates. A cluster of non-haemolytic isolates, one of which was a neonatal invasive disease isolate, appeared to lack the entire cyl locus. Admixture analysis of population structure revealed evidence of genetic transfer among the WA isolates across structural groups. When compared against the PubMLST S. agalactiae data, WA isolates showed high levels of strain diversity with minimal apparent clustering. This is the first whole genome sequence study of WA S. agalactiae isolates and also represents the first addition of Australian isolate data to PubMLST. This report provides insight into the distribution and diversity of vaccine targets of S. agalactiae within Western Australia, indicating that the most appropriate capsular vaccine for this population would be the proposed pentavalent (Cps Ia, Ib, II, III and V) preparation, whilst vaccines targeting surface proteins should ideally utilise Fbp, Lmb and/or ScpB.

New genetic context of lnu(B) composed of two multi-resistance gene clusters in clinical Streptococcus agalactiae ST-19 strains

BackgroundClindamycin is a lincosamide antibiotic used to treat staphylococcal and streptococcal infections. Reports of clinical Streptococcus agalactiae isolates with the rare lincosamide resistance/macrolide susceptibility (LR/MS) phenotype are increasing worldwide. In this study, we characterised three clinical S. agalactiae strains with the unusual L phenotype from China.MethodsThree clinical S. agalactiae strains, Sag3, Sag27 and Sag4104, with the L phenotype were identified from 186 isolates collected from 2016 to 2018 in Shanghai, China. The MICs of clindamycin, erythromycin, tetracycline, levofloxacin, and penicillin were determined using Etest. PCR for the lnu(B) gene was conducted. Whole genome sequencing and sequence analysis were carried out to investigate the genetic context of lnu(B). Efforts to transfer lincomycin resistance by conjugation and to identify the circular form by inverse PCR were made.ResultsSag3, Sag27, and Sag4104 were susceptible to erythromycin (MIC ≤0.25 mg/L) but resistant to clindamycin (MIC ≥1 mg/L). lnu(B) was found to be responsible for the L phenotype. lnu(B) in Sag3 and Sag27 were chromosomally located in an aadE-spw-lsa(E)-lnu(B) resistance gene cluster adjacent to an upstream 7-kb tet(L)-cat resistance gene cluster. Two resistance gene clusters were flanked by the IS6-like element, IS1216. Sag4104 only contained partial genes of aadE-spw-lsa(E)-lnu(B) resistance gene cluster and was also flanked by IS1216.ConclusionThese results established the presence of the L phenotype associated with lnu(B) in clinical S. agalactiae isolates in China. The lnu(B)-containing multi-resistance gene cluster possibly acts as a composite transposon flanked by IS1216 and as a vehicle for the dissemination of multidrug resistance among S. agalactiae.

A cohort study of the effect of Streptococcus agalactiae on milk yield and somatic cell count in Norwegian dairy cows

The primary objective of the present study was to estimate the effect of Streptococcus agalactiae intramammary infection on milk production and somatic cell count (SCC) in Norwegian dairy cows. A secondary objective was to assess differences in the effect of common Strep. agalactiae sequence types (ST) found in Norwegian dairy herds. We performed a cohort study combining registry data with sequence-type data from Strep. agalactiae isolates. Herds in which Strep. agalactiae had been detected in individual animals (bacteriological culture or quantitative PCR) between 2012 and 2015 were included. We accessed monthly test-day milk yield records for the entire period to compare milk yield and SCC between cows that were Strep. agalactiae positive and all other cows, within each herd. The study sample consisted of 150 herds, 15,757 cows, 30,850 lactations, and 204,126 test days. We evaluated the effects of Strep. agalactiae on test-day milk yield and SCC using mixed linear regression models, controlling for clustering by herd, cow, and lactation. Multilocus sequence typing of Strep. agalactiae was available for isolates from 86 herds. Additional models were fit to a subset of herds (n = 59) in which ST1, ST23, ST103, and ST196 had been found, to compare the effects of ST on milk production and SCC. In the period 3 to 2 mo before diagnosis, Strep. agalactiae-positive cows produced an average of 1.3 kg more DIM-adjusted milk/d than their negative herd mates. At the time of diagnosis, production was on average 0.13 kg less DIM-adjusted milk/d in Strep. agalactiae-positive cows than in negative cows; 2 to 3 mo after diagnosis, they produced 1.24 kg less DIM-adjusted milk/d than negative cows. Losses persisted for the rest of the investigated period. Cows with ST23, ST103, and ST196 followed a similar pattern as the overall analysis with respect to milk production, whereas ST1-affected cows produced similar amounts of milk before diagnosis as the negative cows. Cows with ST1 experienced the largest milk loss 1 to 2 mo after diagnosis but then recovered to some extent; for cows with ST103, the severe milk loss persisted for the rest of the investigation period. The cow-associated ST103 elicited a lower response in peak SCC compared with ST23, ST103, and ST196. The results indicate an effect of Strep. agalactiae on milk production and SCC. Production was lowest 2 to 3 mo after a positive sample. Peak SCC was reached the month before diagnosis, with notable differences between sequence types.

Molecular typing of Streptococcus agalactiae isolates of serotype Ia from tilapia in southern China.

Streptococcus agalactiae is an important pathogen of tilapia causing enormous economic losses worldwide. In this study, multilocus sequence typing indicated that 75 S. agalactiae isolates from tilapia in southern China belonged to sequence type-7, as well as belonging to serotype Ia, as confirmed by multiplex PCR assay. The putative-virulence gene profiles and genetic variation of these strains were determined by three sets of multiplex PCR and multi-virulence locus sequencing typing (MVLST), respectively. Analysis of putative-virulence gene profiles showed that each strain harbored 18 putative-virulence genes but lacked lmb and scpB. Three putative-virulence genes (srr-1, bibA and fbsA) were further selected for MVLST analysis. Our data showed that the strains had 14 MVLST types (1-14) and clustered in three groups (Groups I-Ⅲ). The period of time during 2013 and 2014 was an important turning point for the differentiation of the putative-virulence genes of S. agalactiae, as type 1 within Group Ⅱ became the predominant MVLST type. There were significant differences in MVLST types of S. agalactiae isolated from different tilapia farming regions. MVLST assay may improve the discriminatory power and is suitable for understanding the epidemiology of S. agalactiae serotype Ia and screening multivalent vaccine candidate strains.

Molecular serotyping and antimicrobial susceptibility of Streptococcus agalactiae isolated from fish in China

Streptococcus agalactiae (group B streptococcus, GBS) is a major pathogenic bacterium frequently associated with septicemia and meningoencephalitis in fish. In this study, we used molecular typing and the disc diffusion method to determine serotypes and antimicrobial susceptibilities of 28 S. agalactiae isolates from diseased farmed fish (tilapia, Schizothorax prenanti and Schizopygopsis pylzovi) in China. We found that the 28 S. agalactiae isolates belonged only to two serotypes (Ia and III), with serotype Ia being significantly more prevalent than serotype III. Antimicrobial susceptibility results showed that all of the isolates were susceptible to vancomycin, cephalexin and florfenicol. The S. agalactiae isolates examined showed resistance to erythromycin (42.9%), penicillin (35.7%), clindamycin (28.6%), enrofloxacin (35.7%), tetracycline (32.1%), gentamicin (28.6%) norfloxacin (28.6%), ofloxacin (14.3%) and doxycycline (7.14%), but the serotype III isolates showed higher proportions of antibiotic resistance than serotype Ia isolates. The macrolide resistance gene (ermB) was detected in all the 28 isolates and the tetracycline resistance gene (tetM) was detected in 78.6% isolates. All quinolone-resistant S. agalactiae isolates carried mostly double point mutations in DNA with inferred amino acid substitutions including the change of Ser-81 to Leu in the product of gyrA and Ser-79 to Tyr in the product of parC. Our study is a valuable reference for effective antibiotic application of S. agalactiae infections of farmed fish in China, and illustrates the importance of better use of antibiotics to prevent the widespread emergence of resistance S. agalactiae.

A novel bioengineered derivative of nisin displays enhanced antimicrobial activity against clinical Streptococcus agalactiae isolates.

Streptococcus agalactiae is the leading cause of neonatal disease worldwide, and infections caused by this opportunistic pathogen are becoming increasingly more prevalent in adults. With the global incidence of antimicrobial resistance continuing to rise, there is a recognised need for new therapeutic agents. Nisin is a potent antimicrobial peptide with demonstrated broad-spectrum activity against a range of clinically significant pathogens. This study aimed to examine the efficacy of nisin against a clinical population of S. agalactiae isolates and further to investigate the bioactivity of a novel bioengineered derivative of the peptide, designated nisin PV. A deferred antagonism assay was used to assess the bioactivity of wild-type nisin and nisin PV against 122 S. agalactiae isolates. Minimum inhibitory concentrations (MICs) were evaluated to determine the specific activity of both peptides. The genetic basis of nisin resistance among the isolate collection was investigated by PCR detection of the nsr gene. In total, 91.0% (111/122) of the collection showed some level of susceptibility to nisin, whilst 9.0% (11/122) displayed complete resistance. Interestingly, the nisin derivative exhibited enhanced antimicrobial activity for 64.8% of the isolates. The frequency of the nsr gene conferring nisin resistance was 98.4% (120/122), suggesting that resistance may be linked to levels of expression of the protein or other regulatory elements. This study indicates that there is potential for the use of nisin and its derivatives as therapeutic agents against S. agalactiae infections.

Genetic Diversity of Streptococcus agalactiae from Multiple Host Species in China

Background: Streptococcus agalactiae is a serious zoonotic pathogen that causes disease in humans and other animals worldwide. Objectives: Due to the increasing prevalence of S. agalactiae, as well as the transmission risk of S. agalactiae from animals to human, it was important to identify the serotype diversity and molecular typing population structure of S. agalactiae isolated from human, bovine, rabbit, and fish. Methods: Capsular polysaccharide serotypes (CPS), multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE) technologies were used to compare the genetic diversity of 56 S. agalactiae isolates from human, rabbit, bovine, and fish sources in China. Results: We found that two serotypes (Ia, III) existed, with serotype Ia being the most common (85.7%). The 56 isolates were assigned to nine sequence types (STs) and seven clonal complexes (CCs) using MLST. Themost common ST was ST891 (35.7%). The ST19 and ST103 were found in two different isolates, while the remaining STs were found in one isolate. A total of 18 PFGE clusters were identified, however, only cluster P was found in both human and bovine isolates. Conclusions: Our findings suggest that serotypes, STs and PFGE, had no obvious correlation with particular host species. However, the similarity of genotypes of S. agalactiae isolated from different host species, speculating cross-infection possibility between them at genetic level.

Alternative Approaches for Mitigation of Drug Resistance in Streptococcus Species

During infection, S. agalactiae can form biofilms which is associated with increased antimicrobial resistance. Additionally, bacteria when present in a biofilm ,are resistant to various physicochemical stresses. Therefore, this study is aimed to determine the in vitro anti biofilm activities of cinnamon oil and/or silver nanoparticles and augmentin against multidrug resistant S. agalactiae isolates recovered from clinical mastitic dairy cattle. Antimicrobial susceptibility testing of 146 Streptococci isolates revealed that 42.5% (62/146) of Streptococci isolates exhibited a feature of multidrug resistance (MDR), being resistant to more than 3 antibiotics of different classes. Cinnamon oil, silver nanoparticles and augmentin exhibited marked inhibitory activities against S. agalactiae isolates with inhibition zone diameters up to 40 mm, 35 mm and 38 mm respectively. Besides, broth microdilution test of cinnamon oil revealed maximum activities against the tested isolates with minimum inhibitory concentrations (MICs) up to 0.0025 µg/mL. The combination of cinnamon oil and silver nanoparticles combination gave the best result as the MIC values have been reduced to 4 folds. The cinnamon oil alone was capable of reducing the number of cells just after 4 h according to time killing assay. The above results highlight the hopeful antibacterial and antibiofilm features of cinnamon oil and silver nanoparticles in the treatment of emergent resistant S. agalactiae mastitic infections.

Intraspecies Gene Variation within Putative Epitopes of Immunodominant Protein P48 of Mycoplasma agalactiae.

P48 protein of Mycoplasma agalactiae is used to diagnose infection and was identified as potential vaccine candidate. According to the genetic nature of mycoplasma and variable sensitivity in P48-based serological diagnosis tests, intra species variation of P48 nucleotide sequence investigated in 13 field isolates of difference province of Iran along with three vaccine strains. Samples were collected from sheep and goat and were cultured in modified PPLO broth. Two pair of primer employed to confirm genus and species of isolates and a pair of primer has developed to amplify the P48 gene. The sequencing results of PCR products were aligned and analyzed besides published sequences in GenBank. T-Cell and B-Cell epitopes and antigenicity of sequence were computationally predicted. The results have shown P48 nucleotide sequences are 99.9% identical in field isolates and vaccine strain of Iran, but analysis of GenBank published sequences have shown divergence up to 5.3% at the nucleotide level and up to 4.9% divergence in protein level of P48 sequences of Iran isolates and other available sequences in GenBank. Single nucleotide polymorphism exists in 89 positions and variable amino acid was observed at 25 residues. Phylogenetic analyses have shown that Mycoplasma agalactiae isolates fall into three main groups based on P48 nucleotide sequences. Immunoinformatics analysis of all available P48 nucleotide sequences have revealed that gene variation lead to differences in immunological properties, but the gene in Iranian isolates are conservative and stable. The sequence variation in epitopes can be underlying source of antigen heterogeneity as a result, affect serological tests accuracy. Due to the high level of divergence in worldwide isolates and high degree of similarity in P48 protein of Iranian isolates, designing recombinant P48 protein based on local pattern can increase the sensitivity and consistency of serological test.

Mutation in cyl operon alters hemolytic phenotypes of Streptococcus agalactiae

Streptococcus agalactiae infects numerous fish species, causing considerable economic losses during fish cultivation. This study compared the phenotypic differences among S. agalactiae hemolytic variant isolates and investigated the genetic composition of their hemolysin genes. Hemolysin is encoded by the cyl operon and mainly regulated by covS/R, which also regulates encapsulation. In total, 45 S. agalactiae clinical isolates were collected from cultured fishes in Taiwan. Three different hemolytic phenotypes—α, β, and γ—were identified. Of the 45 isolates, 39 were β hemolytic, 3 were α hemolytic, and 3 were γ hemolytic. The γ-hemolytic isolates demonstrated significantly thicker encapsulation and slower growth rates than did the α- and β-hemolytic isolates. However, no isolate had mutations in the regulatory gene covS/R. A 1252-bp insertion sequence (IS) in the cyl operon of α-hemolytic isolates, located at cylF region, was found. This IS interrupted cylF through insertion at 23 bp downstream of starting codon, causing incomplete mRNA transcription. The β-hemolytic isolates showed no mutation in the cyl operon. By contrast, the γ-hemolytic isolates had lost the entire cyl operon; it had been replaced by a 14-kb genomic island containing genes for DNA recombinase and septum formation proteins. In summary, the differences in hemolysin genes between α- and β-hemolytic isolates were due to the IS in the cylF region, whereas in the γ-hemolytic isolates, the entire cyl operon was deleted and replaced. These findings explain different hemolysin expressions of the clinical S. agalactiae isolates taken from fish ponds in Taiwan. ImportanceStreptococcus agalactiae infects both warm- and cold-blooded animals and causes major aquatic cultivation loss. Pathogenic isolates from the outbreak of fish ponds were examined their cyl operon gene. α-Hemolytic isolate with mutant cyl operon was observed for the first time in aquaculture animals and was compared to intact or entire cyl operon deletion of β- and γ-hemolytic isolates. Hemolysis expression levels of Streptococcus agalactiae are explained.