INVESTIGATION OF THE BACTERIA EXTRACTED FROM ERBIL CITY’S SEWAGE WATER AND TREATED WITH CHLORINE

Objective To analyze the clinical features of neonatal purulent meningitis (NPM) caused by Gram-positive and Gram-negative bacteria. Method From January 2008 to December, 2015, the clinical data of NPM with positive cerebrospinal-fluid (CSF) culture admitted to Children′s Hospital of Fudan University were reviewed retrospectively. Patients were assigned into Gram-positive group and Gram-negative group according to CSF culture, and general information, clinical presentation, laboratory examination and outcome were compared between the two groups. Wilcoxon Rank-Sum test was used to compare means. Proportions were compared using χ2 test. Result A total of 82 NPM patients with positive CSF culture were enrolled in the study, and 44 (53.7%) were male. The average gestational age was 38.5 (35.6, 39.6) weeks and the mean birth weight was 3 100 (2 600, 3 380) grams. 16 (19.5%) cases were early-onset meningitis and 66(80.5%) were late-onset. In 82 cases, 43 (52.4%) showed Gram-positive bacteria and 39(47.6%) Gram-negative in CSF culture. The five most common pathogens were escherichia coli(22 cases, 26.8%), group B streptococcus (GBS) (10 cases, 12.2%), enterococcus faecium (8 cases, 9.8%), coagulase-negative staphylococcus(8 cases, 9.8%) and klebsiella pneumoniae(5 cases, 6.1%). In early-onset patients, the main causative pathogens isolated from CSF were GBS (3 cases, 18.8%) and enterococcus (3 cases, 18.8%) . Escherichia coli (20 cases, 30.3%) and coagulase-negative staphylococcus(8 cases, 12.1%) were the most common pathogens in late-onset patients. Gram-positive group and Gram-negative group had similar clinical presentation(P>0.05). The ratio of patients with blood C-reactive protein>8 mg/L in Gram-negative group was higher than that in Gram-positive group(P<0.05). Those with Gram-negative bacterial meningitis had a higher incidence of hydrocephalus than Gram-positive (P<0.05). Conclusion The main pathogens of NPM are escherichia coli and GBS. Neonates with Gram-positive bacteria meningitis have similar clinical presentation with those with Gram-negative meningitis, but have different laboratory examination and complication characteristics. Key words: Meningitis, bacterial; Gram-positive bacteria; Gram-negative bacteria; Clinical features

Group B Streptococcus (GBS) is a species of gram positive bacteria representing a significant human pathogen; namely, as the most prolific cause of neonatal disease and mortality globally, but also increasingly reported in adult disease (especially among the elderly and those with compromised immune systems). The first chapter of this thesis reviews the extensive literature covering GBS; with a focus on classification and disease. Selected virulence factors are also discussed. In chapter 2, eight strains of GBS were selected for whole genome sequencing by Third Generation, Pacific Biosystems (PacBio) sequencing technology. A protocol was optimised to provide sufficient, high quality genomic preparations from multiple strains of GBS – suitable for the PacBio technology. Using a sequenced strain of GBS from chapter 2, an infection model was optimised in chapter 3 for the purpose of providing quality RNA for co-transcript analysis. U937 human monocytes were infected with GBS (strain 874391) and the host/pathogen RNA prepared from the same reaction (monocytes with internalised GBS) – with an emphasis on yielding sufficient pathogen RNA; which can sometime be an impediment for co-transcript studies. Pathogen RNA derived from the optimised infection protocol was demonstrated to amplify with RT-qPCR for 12 tested GBS genes (cylE, 1010, rib, czcD, pil2B, cpsE, scpB, htp, cfb, copA, hvgA and maeA). In chapter 4, RT-qPCR was used to analyse differential gene expression from the mixed, host/pathogen RNA. Twelve human genes and 12 GBS genes were assessed for differential gene expression. Seven of the tested human genes (IL8, IL1A, IL1B, IL10, TNF, LMO2 and MCP-1) and 6 of the tested GBS genes (scpB, 1010, rib, czcD, htp, hvgA) were significantly upregulated in RNA derived from the infection samples. Of the GBS genes tested, htp was the most upregulated. An htp knockout mutant of GBS strain 874391 (Δhtp) was constructed for chapter 5 of this thesis to assess the impact of htp transcription on GBS survival in an infection context. The infection assays optimised in chapter 3 were performed with the Δhtp GBS construct. Contrary to expectation, the Δhtp GBS construct survived the internalised environment of the monocytes in significantly higher numbers than the wild-type over 48 hours of infection.

To study the clinical characteristics, pathogenic bacteria, and antibiotics resistance of neonatal purulent meningitis in order to provide the guide for early diagnosis and appropriate treatment. A retrospective review was performed and a total of 112 cases of neonatal purulent meningitis (male 64, female 58) were identified in the neonatal intensive care unit of Yuying Children's Hospital of Wenzhou Medical University seen from January 1, 2004 to December 31, 2013. The clinical information including pathogenic bacterial distribution, drug sensitivity, head imageology and therapeutic outcome were analyzed. Numeration data were shown in ratio and chi square test was applied for group comparison. Among 112 cases, 46 were admitted from 2004 to 2008 and 66 from 2009 to 2013, 23 patients were preterm and 89 were term, 20 were early onset (occurring within 3 days of life) and 92 were late onset meningitis (occurring after 3 days of life). In 62 (55.4%) cases the pathogens were Gram-positive bacteria and in 50 (44.6%) were Gram-negative bacteria. The five most frequently isolated pathogens were Escherichia coli (32 cases, 28.6%), coagulase-negative staphylococcus (CNS, 20 cases, 17.9%), Streptococcus (18 cases, 16.1%, Streptococcus agalactiae 15 cases), Enterococci (13 cases, 11.6%), Staphylococcus aureus (9 cases, 8.0%). Comparison of pathogenic bacterial distribution between 2004-2008 and 2009-2013 showed that Gram-positive bacteria accounted for more than 50% in both period. Escherichia coli was the most common bacterium, followed by Streptococcus in last five years which was higher than the first five years (22.7% (15/66) vs. 6.5% (3/46), χ(2) = 5.278, P < 0.05). Klebsiella pneumoniae was more common isolate in preterm infants than in term infants (13.0% (3/23) vs. 1.1% (1/89), χ(2) = 7.540, P < 0.05). Streptococcus (most were Streptococcus agalactiae) was the most common bacteria in early onset meningitis and higher than those in late onset meningitis (35.0% (7/20) vs. 12.0% (11/92), χ(2) = 4.872, P < 0.05). Drug sensitivity tests showed that all the Gram-positive bacterial isolates were sensitive to linezolid. Staphylococci were resistant to penicillin, and most of them were resistant to erythromycin, oxacillin and cefazolin; 77.8%of CNS isolates were methicillin-resistant staphylococcus. No Streptococcus and Enterococcus faecalis was resistant to penicillin. None of enterococci was resistant to vancomycin. Among the Gram-negative bacterial isolates, more than 40% of Escherichia coli were resistant to commonly used cephalosporins such as cefuroxime, cefotaxime and ceftazidime, and all of them were sensitive to amikacin, cefoperazone sulbactam and imipenem. Isolates of Klebsiella pneumoniae were all resistant to ampicillin, cefuroxime, cefotaxime and ceftazidime, but none of them was resistant to piperacillin tazobactam and imipenem. Of the 112 patients, 69 were cured, 23 improved, 9 uncured and 11 died. There were 47 cases (42.0%) with poor prognosis, they had abnormal head imageology, severe complications and some cases died, 13 of 18 (72.2%) patients with meningitis caused by Streptococcus died. Escherichia coli, CNS and Streptococcus are the predominant pathogens responsible for neonatal purulent meningitis over the past ten years. There were increasing numbers of cases with Streptococcus meningitis which are more common in early onset meningitis with adverse outcome, therefore careful attention should be paid in clinic. Linezolid should be used as a new choice in intractable neonatal purulent meningitis cases caused by gram positive bacteria.

Group B Streptococcus (GBS) is one of the important pathogens in adverse effects on pregnancy outcomes. GBS is also the leading cause of complications such as neonatal sepsis and meningitis and death of perinatal infants. There are ten different serotypes of GBS infection. GBS screening for pregnant women in perinatal period can provide references for clinicians taking intrapartum antibacterial prophylaxis (IAP) measures timely on pregnant women to prevent perinatal GBS infection, and effectively reduce infection rate of perinatal GBS. As a simple and safe method to prevent GBS infection, GBS vaccines have been paid more and more attention. GBS vaccines are currently the most prospective measure to prevent neonatal GBS infection through maternal immunity, but they have no preventive effects on neonatal-late onset GBS infection. This article focuses on adverse pregnancy outcomes, risk factors, effects of regional differences, diagnosis, treatment caused by perinatal GBS infection and current research development of GBS vaccines. Key words: Streptococcus agalactiae; Infectious disease transmission, vertical; Sepsis, neonatal; Income, socioeconomic factors, family characteristics; Pregnancy outcome; Vaccination, maternal

Objective To raise awareness of the late-onset meningitis caused by group B streptococcus (GBS) which was homogenous to the maternal colonization. Methods The clinical data of late-onset GBS meningitis in neonates twins whose pathogens were homogenous to their maternal colonization were collected from Department of Neonatology, Guangzhou Women and Children′s Medical Center.The general conditions, clinical symptoms, laboratory tests and drug treatment of the twins and their mother were retrospectively analyzed, and the GBS homology during inpatient care was tested.And the progress of the twins′ condition was investigated by telephone follow-up. Results The mother had two pregnancies without prenatal GBS screening or intrapartum antimicrobial intervention for GBS, everything was normal during pregnancy and delivery.Twins were born through cesarean section.The elder sister was discharged with Linezolid taken orally after 167 days in hospital without convulsions, shaking or other discomfort.The elder sister was followed up for every 2 weeks, and in the last time of follow-up, cerebrospinal fluid white blood cell counts were 45×106/L, protein level was 1.52 g/L and Linezolid was withdrawn.The younger brother was discharged after 58 days in hospital with follow-up for every 2 weeks, and in the last time of follow-up, cerebrospinal fluid white blood cell counts were 30×106/L, protein level was 0.66 g/L.During the hospitalization and follow-up without convulsions and irritation, and the cranial magnetic resonance imaging of the twin brother was normal.Test results showed that the GBS bacteria strain for twins and their mother were all serotype Ⅲ.The possibility of the GBS homology was more than 90%. Conclusions The toxicity of serotype Ⅲ GBS strain was strong.More proactive precautions should be considered to apply for the mother whose first birth already had GBS infection.Early identification and intervention of infection risk factors would help optimize the anti-infection treatment program and reduce nerve system damage and other adverse outcomes caused by invasive GBS infection. Key words: Group B streptococcus; Infant, newborn; Meningitis

With growing concerns about Group B streptococcal (GBS) infections and their adverse effects on perinatal pregnancies, including infection, premature delivery, neonatal septicemia, and meningitis, it is urgent to promote GBS screening at all pregnancy stages. The purpose of this study is to establish a device-independent, fast, sensitive, and visual GBS detection method. Taking advantage of the characteristics of the recombinase polymerase isothermal amplification (RPA), the activity of the nfo nuclease cleavage base analog (tetrahydrofuran, THF) site, and the advantages of visual reading of the lateral flow chromatography strip (LFS), a GBS detection method was developed. This method focused on the conservative region of the Christie-Atkins-Munch-Petersen factor encoded by the cfb gene, a virulence gene specific to GBS. Two forward primers, two biotin-labeled reverse primers, and one fluorescein isothiocyanate (FITC)-labeled and C3spacer-blocked probe were designed. The study involved optimizing the primer pair and probe combination, determining the optimal reaction temperature and time, evaluating specificity, analyzing detection limits, and testing the method on 87 vaginal swabs from perinatal pregnant women. The results showed that the visual detection method of GBS-RPA-LFS, using the cfb-F1/R2/P1 primer probe, could detect GBS within 15 min at the temperature ranging from 39°C to 42°C. Furthermore, the method specifically amplified only GBS, without cross-reacting with pathogens like Lactobacillus iners, Lactobacillus crispatus, Candida albicans, Listeria monocytogenes, Yersinia enterocolitica, Klebsiella Pneumoniae, Enterobacter cloacae, Citrobacter freundii, Vibrio alginolyticus, Vibrio parahaemolyticus, Salmonella typhimurium, Staphylococcus aureus, Pseudomonas aeruginosa, or Trichomonas vaginalis. It could detect a minimum of 100 copies per reaction. In clinical 98 samples of vaginal swabs from pregnant women, the agreement rate between the GBS-RPA-LFS method and TaqMan real-time fluorescence quantification method was 95.92%. In conclusion, this study successfully established a combined RPA and LFS GBS in situ detection platform, with short reaction time, high sensitivity, high specificity, portability, and device independence, providing a feasible strategy for clinical GBS screening.

Group B Streptococcus (GBS) is one of the important pathogens in the infection of perinatal neonates. GBS has higher rate of morbidity and mortality in GBS infection of perinatal neonates. With the application of prophylactic antibiotics during labor, the rate of GBS infection in perinatal neonates has significantly decreased, but the rate is currently in stable period. There are ten different serum types of GBS infection. We want to elaborate the characteristics of GBS infection of perinatal neonates from biological, epidemiological and different serum types, and prevention strategies of GBS infection, in order to enhance the knowledge of different serum types of GBS infection in neonatal for clinicians, to find more effective methods or strategies to further reduce the incidence of GBS infection of perinatal neonates, and provide a theoretical basis for decreasing the rate of treatment by antibiotics. Key words: Streptococcus agalactiae; Serological typing; Perinatal mortality; Universal precautions; Streptococcal infections; Infant, newborn

Objective To investigate the clinical characteristics, prenatal risk factors and antibiotic resistance of neonatal early-onset blood stream infection caused by group B Streptococcus (GBS) and to provide evidence for the management of GBS infection. Methods From January 1, 2001 to May 31, 2017, 55 neonates diagnosed with GBS-associated early-onset blood stream infection in the International Peace Maternity & Child Health Hospital were enrolled. They were divided into two groups: GBS early-onset sepsis (GBS EOS) group (n=40) and bacteremia group (n=15). Clinical data were retrospectively analyzed and compared between the two groups with two independent-samples t test and Chi-square test. Results The incidence of neonatal GBS-associated early-onset blood stream infection was 0.31‰(55/179 738). Those neonates with bacteremia had no clinical manifestations. However, in the GBS EOS group, the main manifestations were respiratory abnormalities [respiratory distress (35%, 14/40) and apnea (10%, 4/40)], jaundice (40%, 16/40) and poor perfusion [low blood pressure, etc. 32%(13/40)], and the main abnormal results of blood routine test were increased C-reaction protein [>10 mg/L, 100% (40/40)] and elevated white blood cell count [>25×109/L, 78% (31/40)]. Compared with GBS EOS group, bacteremia group had a lower rate of vaginal delivery [1/15 vs 52% (21/40), χ2=9.549, P=0.002] and higher rates of maternal fever [15/15 vs 55% (22/40), χ2=10.034, P=0.002], chorioamnionitis [15/15 vs 68% (27/40), χ2=6.384, P=0.012] and empirical administration of antibiotics to infants [15/15 vs 70% (28/40), χ2=5.759, P=0.016]. All tested GBS strains were susceptible to penicillin, ampicillin and vancomycin, but resistant to clindamycin (42%, 22/53) and erythromycin (46%, 24/52). Conclusions GBS is one of the predominant pathogens causing neonatal early-onset blood stream infection. Respiratory distress, apnea, jaundice and poor perfusion are the main manifestations. Penicillin and ampicillin are the treatment choice for neonatal early-onset GBS infection. Active and appropriate intrapartum management of pregnancies with high risk of intrauterine GBS infection as well as prophylactic prescription of antibiotics to infants may help to prevent or alleviate the symptoms of neonatal early-onset blood stream infection. Key words: Bacteremia; Streptococcus agalactiae; Streptococcal infections; Microbial sensitivity tests; Infant, newborn

Group B Streptococcal (GBS), a gram-positive Streptococcus, is one of the important pathogenic bacteria which can causes neonatal sepsis and meningitis. The main measure of GBS is intrapartum antibiotics prevention (IAP), which plays an important role in early-onset GBS disease (GBS-EOD), but remains a high infection rates in late-onset GBS disease(GBS-LOD). What′s more, with the extensive use of antimicrobial drugs, the GBS drug resistance rate increased gradually. So that GBS vaccines provide a new method to prevent neonatal GBS infection, especially the capsular polysaccharide conjugate vaccine, the phase Ⅰ and Ⅱ clinical trials have been completed, and the protective antibody levels has been researching. This research will focus on the research progress on GBS vaccines in order to provide reference for further development of GBS vaccines. Key words: Streptococcus agalactiae; Infection; Vaccines; Antibodies

Group B Streptococcus (GBS) is an important pathogen which may result in miscarriage, intrauterine infection and puerperal infection. Neonatal GBS infection may lead to septicemia, pneumonia and meningitis. GBS can be divided into a variety of serotypes according to the antigenic structure of capsular polysaccharide, and different serotypes of GBS vary in ethnicity, geographical distribution, pathogen virulence and pathogenic species. The distribution and identification of GBS serotypes, and their relationships with genotypes, drug resistance, virulence factors and vaccine preparation of GBS were reviewed. Key words: Peripartum period; Streptococcus agalactiae; Streptococcal infections; Serotyping

Objective To investigate the incidence and pathogen distribution of neonatal early-onset sepsis (EOS) following exposure to antenatal antibiotics. Methods One hundred and eighty-four neonates who were admitted to Maternal and Child Care Hospital of Xiamen and identified as having EOS from January 2010 to December 2015 were enrolled. The clinical data were retrospectively analyzed. According to antenatal antibiotic exposure time, the infants were divided into the antibiotics group (≥4 hours) and the control group (<4 hours). Women in late pregnancy (35-37 weeks of gestation) underwent group B Streptococcus (GBS) screening using standard bacterial culture beginning from Janaury 2014 as screening group. Intrapartum antibiotic prophylaxis was given if the GBS culture was positive. Infants delivered before January 2014 were included in the no-screening group. Pathogen distribution and the difference in drug resistance between the two groups were compared by a two-independent samples t-test and Chi-square test. Results In the antibiotics group, the percentages of birth weight lower than 2 500 g, preterm infants, asphyxia, and positive rates of GBS and blood culture were 24.3%(17/70), 14.3% (10/70), 2.9% (2/70), 7.1% (5/70) and 70.0% (49/70), respectively, and were significantly lower than those in the control group [39.5%(45/114), 28.1% (32/114), 14.9% (17/114), 19.3% (22/114) and 88.6% (101/114), respectively] (χ2=4.478, 4.678, 6.807, 5.118 and 9.957, all P 0.05). Compared with the no-screening group, the positive rate of GBS decreased [7.6% (5/66) vs 18.6% (22/118)] and the positive rate of fungal infection increased [7.6% (5/66) vs 1.7% (2/118)] in the screening group (χ2=4.141, P=0.042; χ2=4.000, P=0.046). The distribution of other pathogenic bacteria such as coagulase-negative Staphylococci and E. coli was not significantly different between the two groups (P>0.05, respectively). Drug resistance rates of Staphylococcus (Staphylococcus aureus and coagulase-negative Staphylococcus) to oxacillin and piperacillin-sulbactam were higher in the screening group than in the no-screening group [82.6% (19/23) vs 52.9% (18/34), χ2=5.302; 78.3% (18/23) vs 47.1% (16/34), χ2=5.549; both P<0.05], and no vancomycin resistant bacterial strains were found. Conclusions Antenatal antibiotic exposure may be effective in reducing the occurrence of prematurity, asphyxia,and GBS infection, but it increases the rate of fungal infection, and is not effective in reducing the incidence of complications and mortality or in changing the distribution of the other pathogens in EOS. Rational indications and timing of antenatal antibiotic exposure should be taken into consideration to reduce drug resistance. Key words: Sepsis; Antibiotic prophylaxis; Prenatal care; Infant, newborn

Objective To research the risk factors and bacterial spectrum of early-onset stroke-associated pneumonia (EOP).Method Retrospectively analysis was conducted to study the clinical material of 54 patients with EOP and 50 patients with non-EOP stroke from Jan 2008 to Dec 2010.Results There were significant differences of smoke,diabetes,pulmonary disease,dystrophy,vomit,aspiration,stroke position and range,National Institute of Health Stroke Scale (NIHSS),consciousness disorder,dysphasia,nasogastric feeding,trachea cannula,tracheotomy and craniotomy between the group of EOP and control group (P＜0.01).There were no significant differences of medicine and position of patients (P＞0.05).There were 8 cases infected with pseudomonas aeruginosa,5 cases with escherichia coli,7 cases with klebsiella pneumonia,3 cases with acinetobacter baumannii,4 cases with staphylococcus aureus,8 cases with proteus mirabilis,6 cases with enterobacter cloacae,2 cases with streptococcus agalactia,3 cases with albicans saccharomyces,2 cases with Candida tropicalis,2 cases with enterococcus faecium,2 cases with streptococcus A,2 cases with Neisseria.Conclusion The pathogen of EOP is community acquired pathogens or hospital acquired pneumonia pathogen with potential drug resistance.The critical factors are weakening of resistance power,planting of bacterium and aspiration.The correct measures should include nutritional support,respiratory management,reduce of bacterial dystopy and reasonable use of antibiotic. Key words: Early-onset pneumonia (EOP) ; Stroke-associated pneumonia (SAP) ; Stroke; Risk factor; Pathogen

Group B Streptococcus (GBS) is a Gram-positive human pathogen representing one of the most common causes of life-threatening bacterial infections such as sepsis and meningitis in neonates. Covalently polymerized pilus-like structures have been discovered in GBS as important virulence factors as well as vaccine candidates. Pili are protein polymers forming long and thin filamentous structures protruding from bacterial cells, mediating adhesion and colonization to host cells. Gram-positive bacteria, including GBS, build pili on their cell surface via a class C sortase-catalyzed transpeptidation mechanism from pilin protein substrates that are the backbone protein forming the pilus shaft and two ancillary proteins. Also the cell-wall anchoring of the pilus polymers made of covalently linked pilin subunits is mediated by a sortase enzyme. GBS expresses three structurally distinct pilus types (type 1, 2a and 2b). Although the mechanisms of assembly and cell wall anchoring of GBS types 1 and 2a pili have been investigated, those of pilus 2b are not understood until now. Pilus 2b is frequently found in ST-17 strains that are mostly associated with meningitis and high mortality rate especially in infants. In this work the assembly mechanism of GBS pilus type 2b has been elucidated by dissecting through genetic, biochemical and structural studies the role of the two pilus-associated sortases. The most significant findings show that pilus 2b assembly appears “non-canonical”, differing significantly from current pilus assembly models in Gram-positive pathogens. Only sortase-C1 is involved in pilin polymerization, while the sortase-C2 does not act as a pilin polymerase, but it is involved in cell-wall pilus anchoring. Our findings provide new insights into pili biogenesis in Gram-positive bacteria. Moreover, the role of this pilus type during host infection has been investigated. By using a mouse model of meningitis we demonstrated that type 2b pilus contributes to pathogenesis of meningitis in vivo.

Effective strategies to prevent infant death rely on knowledge of prevalent pathogens. Recent publications have drawn attention to limited data on the contribution of group B streptococcal infection to neonatal and infant mortality in resource-restricted settings. The aim was to describe all cases of group B streptococcal infection isolated from the blood culture of infants up to 90 days of age in two South African hospitals over a two-year period. A retrospective record review took place of infants in whom group B streptococcus was isolated from blood culture or cerebrospinal fluid from January 2010 and December 2011. The maternal records of infants were also reviewed. Data were analysed using Stata® version 1.1. Forty-one cases of group B streptococcal infection were identified, for which 33 records were available for analysis. There was early-onset disease in 14 (42.4%) and late-onset disease in 19 (57.6%) of the infants. Eight (24%) infants were human immunodeficiency virus (HIV)- exposed. There was a...

Group B streptococcus infection induces trophoblast apoptosis