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Ethoxzolamide (EZA), acetazolamide, and methazolamide are clinically used sulphonamide drugs designed to treat non-bacteria-related illnesses (e.g. glaucoma), but they also show antimicrobial activity against the gastric pathogen Helicobacter pylori. EZA showed the highest activity, and was effective against clinical isolates resistant to metronidazole, clarithromycin, and/or amoxicillin, suggesting that EZA kills H. pylori via mechanisms different from that of these antibiotics. The frequency of single-step spontaneous resistance acquisition by H. pylori was less than 5 × 10−9, showing that resistance to EZA does not develop easily. Resistance was associated with mutations in three genes, including the one that encodes undecaprenyl pyrophosphate synthase, a known target of sulphonamides. The data indicate that EZA impacts multiple targets in killing H. pylori. Our findings suggest that developing the approved anti-glaucoma drug EZA into a more effective anti-H. pylori agent may offer a faster and cost-effective route towards new antimicrobials with a novel mechanism of action.

During its intraerythrocytic life cycle, the human malaria parasite Plasmodium falciparum supplements its nutritional requirements by scavenging substrates from the plasma through the new permeability pathways (NPPs) installed in the red blood cell (RBC) membrane. Parasite proteins of the RhopH complex: CLAG3, RhopH2, RhopH3, have been implicated in NPP activity. Here we studied 13 exported proteins previously hypothesised to interact with RhopH2, to study their potential contribution to the function of NPPs. NPP activity assays revealed that the 13 proteins do not appear to be individually important for NPP function, as conditional knockdown of these proteins had no effect on sorbitol uptake. Intriguingly, reciprocal immunoprecipitation assays showed that five of the 13 proteins interact with all members of the RhopH complex, with PF3D7_1401200 showing the strongest association. Mass spectrometry-based proteomics further identified new protein complexes; a cytoskeletal complex and a Maurer's clefts/J-dot complex, which overall helps clarify protein-protein interactions within the infected RBC (iRBC) and is suggestive of the potential trafficking route of the RhopH complex itself to the RBC membrane. This article is protected by copyright. All rights reserved.

With the rise of bacterial resistance to conventional antibiotics, re-purposing of Food and Drug Administration (FDA) approved drugs currently used to treat non-bacteria related diseases as new leads for antibacterial drug discovery has become an attractive alternative. Ethoxzolamide (EZA), an FDA-approved diuretic acting as a human carbonic anhydrase inhibitor, is known to kill the gastric pathogenic bacterium Helicobacter pylori in vitro via an, as yet, unknown mechanism. To date, EZA activity and resistance have been investigated for only one H. pylori strain, P12. We have now performed a susceptibility and resistance study with H. pylori strains SS1 and 26695. Mutants resistant to EZA were isolated, characterized and their genomes sequenced. Resistance-conferring mutations were confirmed by backcrossing the mutations into the parent strain. As with P12, resistance to EZA in strains SS1 and 26695 does not develop easily, since the rate of spontaneous resistance acquisition was less than 10−8. Acquisition of resistance was associated with mutations in 3 genes in strain SS1, and in 6 different genes in strain 26695, indicating that EZA targets multiple systems. All resistant isolates had mutations affecting cell wall synthesis and control of gene expression. EZA’s potential for treating duodenal ulcers has already been demonstrated. Our findings suggest that EZA may be developed into a novel anti-H. pylori drug.

Additional file 1: Table S1. Nucleotide changes in the H. pylori SS1 mutant resistant to EZA.

α-Carbonic anhydrase of Helicobacter pylori (HpαCA) plays an important role in the acclimation of this oncobacterium to the acidic pH of the stomach. Sulfonamide inhibitors of HpαCA possess anti-H. pylori activity. The crystal structures of complexes of HpαCA with a family of acetazolamide-related sulfonamides have been determined. Analysis of the structures revealed that the mode of sulfonamide binding correlates well with their inhibitory activities. In addition, comparisons with the corresponding inhibitor complexes of human carbonic anhydrase II (HCAII) indicated that HpαCA possesses an additional, alternative binding site for sulfonamides that is not present in HCAII. Furthermore, the hydrophobic pocket in HCAII that stabilizes the apolar moiety of sulfonamide inhibitors is replaced with a more open, hydrophilic pocket in HpαCA. Thus, our analysis identified major structural features can be exploited in the design of selective and more potent inhibitors of HpαCA that may lead to novel antimicrobials.

Group B Streptococcus (GBS) is a predominant cause of early-onset neonatal sepsis globally; however, the impact of this organism on the health of newborns in South Asia is debated, due in part to a paucity of community-based assessments. We estimated the prevalence and serotypes of GBS colonization of the umbilical cord stump and the association of colonization with mortality in a population-based cohort of newborns in rural Sylhet District, Bangladesh.Umbilical cord swabs were collected from 646 newborns up to 3 times within the first week after birth (ages24 hours, ~3 days, ~6 days) and processed to identify GBS serotypes.GBS was isolated from 6.3% (41/646) of newborns. Sixty-one percent of the GBS colonization occurred in neonates within 24 hours of delivery. Serotypes VII (37.1%, n = 13/36) and Ia (33.3%, n = 12/36) were the most predominant colonizing GBS isolates. Other detected serotypes were Ib (11.1%, n = 4/36), II (11.1%, n = 4/36), V (5.6%, n = 2/36) and VI (2.8%, n = 1/36). Mortality risk among newborns with GBS colonization was 6.6 (95% confidence interval: 2.1-20.4) times higher than for those without GBS.The overall prevalence of GBS colonization was lower than in settings, where GBS is a predominant etiology of neonatal illness. In addition, the GBS serotype distribution differed from that reported in the developed part of the world. However, further studies are needed to understand the true burden of GBS-related illness. Mortality risk was substantially increased in the presence of GBS on the umbilical stump, providing support for chlorhexidine antisepsis to the cord to prevent colonization of invasive pathogens.

Global swine populations infected with influenza A viruses pose a persistent pandemic risk. With the exception of a few countries, our understanding of the genetic diversity of swine influenza viruses is limited, hampering control measures and pandemic risk assessment. Here we report the genomic characteristics and evolutionary history of influenza A viruses isolated in Australia during 2012–2016 from two geographically isolated swine populations in the states of Queensland and Western Australia. Phylogenetic analysis with an expansive human and swine influenza virus dataset comprising >40,000 sequences sampled globally, revealed evidence of the pervasive introduction and long-term establishment of gene segments derived from several human influenza viruses of past seasons, including H1N1/1977, H1N1/1995, H3N2/1968, H3N2/2003, and H1N1pdm09, and a genotype that contained gene segments derived from the past three pandemics (1968, re-emerged 1977 and 2009). Of the six human-derived gene lineages only one comprising two viruses isolated in Queensland during 2012 was closely related to swine viruses detected from other regions, indicating a previously undetected circulation of Australian swine lineages for approximately 3–44 years. Although the date of introduction of these lineages into Australian swine populations could not be accurately ascertained, we found evidence of sustained transmission of two lineages in swine during 2012–2016. The continued detection of human-origin influenza virus lineages in swine over several decades with little or unpredictable antigenic drift indicates that isolated swine populations can act as ‘antigenic archives’ of human influenza, raising the risk of re-emergence in humans when sufficient susceptible populations arise.IMPORTANCE We described the evolutionary origins and antigenic properties of influenza A viruses isolated from two separate Australian swine populations during 2012–2016, showing that these viruses were distinct to each other and to those isolated from swine globally. Whole genome sequencing of virus isolates revealed a high genotypic diversity that had been generated exclusively through the introduction and establishment of human influenza viruses that circulated in past seasons. We detected six reassortants with gene segments derived from human H1N1/H1N1pdm09 and various human H3N2 viruses that circulated at various periods since 1968. We also found that these swine viruses were not related to swine viruses collected elsewhere indicating independent circulation. The detection of unique lineages and genotypes in Australia suggests that isolated swine populations that are sufficiently large can sustain influenza for extensive periods, we showed direct evidence of a sustained transmission for at least 4 years between 2012–2016.

Detection of pneumococcal carriage by multiple co-colonizing serotypes is important in assessing the benefits of pneumococcal conjugate vaccine (PCV). Various methods differing in sensitivity, cost and technical complexity have been employed to detect multiple serotypes of pneumococcus in respiratory specimens. We have developed an algorithmic method to detect all known serotypes that preserves the relative abundance of specific serotypes by using Quellung-guided molecular techniques. The method involves culturing respiratory swabs followed by serotyping of 100 colonies by either capsular (10 colonies) or PCR (90 colonies) reactions on 96-well plates. The method was evaluated using 102 nasal swabs from children carrying pneumococcus. Multiple serotypes were detected in 22% of carriers, compared to 3% by World Health Organization (WHO)-recommended morphology-based selection of 1 to 3 colonies. Our method, with a processing cost of $87, could detect subdominant strains making up as low as 1% of the population. The method is affordable, practical, and capable of detecting all known serotypes without false positive reactions or change in the native distribution of multiple serotypes.

Group B streptococcus (GBS) infection is a leading cause of death among newborns in developed countries. Data on the burden of GBS in Asian countries are lacking. This study aimed to understand (i) the rate of maternal rectovaginal GBS carriage, (ii) the rate of vertical transmission of GBS, as determined by culturing ear, umbilicus, and nasal swabs, and (iii) the distribution of GBS serotypes. This prospective observational study was conducted between September 2012 and November 2013 at Kumudini Women's Medical College Hospital, a secondary-level hospital in Mirzapur, Bangladesh. The study enrolled pregnant women who visited the outpatient clinic for antenatal care (ANC) and/or delivered a child in the inpatient department of Kumudini Women's Medical College Hospital and the babies born to those mothers. Among 1,151 enrolled pregnant women, 172 (15% [95% confidence interval [CI], 13 to 17%]) carried GBS; among 68 babies born to mothers with carriage, 26 (38% [95% CI, 27 to 51%]) had GBS on their body surfaces, indicating vertical transmission. Typing of the isolates ( n = 172) identified all 10 GBS serotypes, most commonly types Ia (40% [69/172 isolates]), V (23% [40/172 isolates]), II (14% [24/172 isolates]), and III (12% [20/172 isolates]). This study shows that Bangladesh has all of the ingredients for invasive GBS disease, including colonization of mothers by invasive serotypes and vertical transmission to babies.

To estimate the prevalence of maternal colonizers in South Asia and their potential to colonize the umbilicus, an important precondition causing neonatal sepsis. We conducted a cross-sectional study at a maternity center in Dhaka with 1219 pregnant women and a subset of 152 newborns from 15 January to 31 October 2011. During labor, study paramedics collected vaginal swabs for bacterial culture and rectal swabs for Group B Streptococcus (GBS) testing. Community health workers collected neonatal umbilical swabs. Log-binomial regression models were used to estimate risk ratios. In all, 454 women (37.2%, 95% confidence interval (CI) 34.5 to 40.0%) were colonized. The most common organisms isolated were Staphylococcus aureus, Non-GBS and GBS. A total of 94 women (7.7%, 95% CI 6.2 to 9.2%) were colonized with GBS. The risk of GBS umbilical colonization was higher (RR=12.98, 95% CI 3.97 to 42.64) among newborns of mothers with GBS colonization. Newborns of mothers colonized with GBS are at higher risk of developing umbilical colonization.

Helicobacter pylori is an important human pathogenic bacterium associated with numerous severe gastroduodenal diseases, including ulcers and gastric cancer. Cytosolic leucyl aminopeptidase (LAP) is an important housekeeping protein that is involved in peptide and protein turnover, catabolism of proteins and modulation of gene expression. LAP is upregulated in metronidazole-resistant H. pylori, which suggests that, in addition to having an important housekeeping role, LAP contributes to the mechanism of drug resistance. Crystals of H. pylori LAP have been grown by the hanging-drop vapour-diffusion method using polyethylene glycol as a precipitating agent. The crystals belonged to the primitive triclinic space group P1, with unit-cell parameters a = 97.5, b = 100.2, c = 100.4 Å, α = 75.4, β = 60.9, γ = 81.8°. An X-ray diffraction data set was collected to 2.8 Å resolution from a single crystal. Molecular-replacement results using these data indicate that H. pylori LAP is a hexamer with 32 symmetry.

Objective To estimate the risk of early-onset neonatal sepsis among newborns of mothers with chorioamnionitis and/or bacterial colonisation in Dhaka. Methods We conducted a cohort study at a maternity centre following 600 mother–newborn pairs. Women with a positive bacterial vaginal culture or positive Group B streptococcus (GBS) rectal culture during labour were classified as colonised. Women with placental histopathology demonstrating signs of maternal or foetal inflammation were classified as having chorioamnionitis. Newborns were followed over the first 7 days of life. The primary outcome measure was physician or community health worker diagnosis of neonatal sepsis following modified World Health Organization Integrated Management of Childhood Illnesses criteria. Survival analysis was conducted with non-parametric, parametric and semiparametric models. Results Of the 600 mother–newborn pairs, 12.8% of newborns were diagnosed with early-onset sepsis. Five hundred and forty-three women had both colonisation and chorioamnionitis data, 55.4% of mothers were non-exposed, 31.7% were only colonised and 12.9% had chorioamnionitis regardless of colonisation status. After adjusting for birthweight, sex, maternal characteristics and wealth, newborns of only colonised mothers developed sepsis 63% faster and had a 71% higher risk of developing sepsis than their non-exposed counterparts (RT = 0.37, 95% CI 0.14–1.03; RH = 1.71, 95% CI 1.00–2.94). Newborns of mothers with chorioamnionitis developed sepsis 74% faster and had a 111% higher risk of developing sepsis (RT = 0.26, 95% CI 0.07–0.94; RH = 2.11, 95% CI 1.06–4.21). Conclusions Newborns born to mothers with colonisation or chorioamnionitis developed sepsis faster and were at higher risk of developing sepsis in Dhaka. Objectif Estimer le risque de septicemie neonatale precoce chez les nouveau-nes de meres atteintes d'une chorioamnionite et/ou d'une colonisation bacteriennes a Dhaka. Methodes Etude de cohorte dans un centre de maternite sur 600 paires mere/nouveau-ne. Les femmes ayant une culture bacterienne vaginale positive ou une culture GBS rectale positive au cours du travail ont ete classees comme colonisees. Les femmes avec une histopathologie placentaire demontrant des signes d'inflammation de la mere ou du fœtus ont ete classees comme ayant une chorioamnionite. Les nouveau-nes ont ete suivis au cours des sept premiers jours de leur vie. Le critere principal mesure etait le diagnostic de la septicemie neonatale par un medecin ou un agent de sante communautaire selon les criteres modifies de la ‘Gestion Integree des Maladies Infantiles de l’OMS’. L'analyse de survie a ete realisee avec des modeles non parametriques, parametriques et semi-parametriques. Resultats Sur les 600 paires mere/nouveau-ne, 12,8% des nouveau-nes ont ete diagnostiques avec une septicemie precoce. 543 femmes avaient a la fois une colonisation et des resultats de chorioamnionite. 55,4% des meres etaient non exposees, 31,7% d'entre elles etaient seulement colonisees et 12,9% avaient une chorioamnionite independamment de l’etat de la colonisation. Apres ajustement pour le poids de naissance, le sexe, les caracteristiques maternelles et la richesse, les nouveau-nes de meres seulement colonisees ont developpe des sepsis 63% plus rapidement et avaient un risque 71% plus eleve de developper une septicemie que leurs homologues non exposes (RT = 0,37; IC95%: 0,14 a 1,03; HR = 1,71; IC95%: 1,00 a 2,94). Les nouveau-nes de meres avec une chorioamnionite ont developpe une septicemie 74% plus vite et avaient un risque 111% plus eleve de developper une septicemie (RT = 0,26; IC95%: 0,07 a 0,94; HR = 2,11; IC95%: 1,6 a 4,21). Conclusions Les nouveau-nes nes de meres avec une colonisation ou une chorioamnionite developpaient une septicemie plus rapidement et etaient a risque plus eleve de developper une septicemie a Dhaka. Objetivo Calcular el riesgo de aparicion temprana de sepsis neonatal entre recien nacidos de madres con corioaminionitis y/o colonizacion bacteriana en Dhaka. Metodos Estudio de cohortes en una maternidad en el que se siguio a 600 parejas madre-neonato. Se clasificaron como colonizadas aquellas mujeres con un cultivo vaginal positivo para bacterias o un cultivo rectal positivo para estreptococo del grupo B durante el parto. Las mujeres con una histopatologia de la placenta con signos de inflamacion materna o fetal fueron clasificadas como padeciendo corioamnionitis. Los neonatos se siguieron durante sus primeros siete dias de vida. El parametro de interes primario era el diagnostico del medico o trabajador sanitario comunitario sobre la sepsis neonatal, siguiendo los criterios de la OMS para la Atencion Integrada a las Enfermedades Prevalentes en la Infancia (AIEPI). Se realizo un analisis de supervivencia con modelos no parametricos, parametricos y semiparametricos. Resultados A un 12.8% de los neonatos, pertenecientes a las 600 parejas madre-neonato, se les diagnostico sepsis de aparicion temprana. 543 mujeres tenian datos tanto de colonizacion como de corioamnionitis. Un 55.4% de las mujeres no estaban expuestas, 31.7% estaban solo colonizadas y 12.9% tenian corioamnionitis, independientemente del estatus de colonizacion. Despues de ajustar para peso al nacer, el sexo, las caracteristicas maternas y el nivel de riqueza, los neonatos de madres solo colonizadas desarrollaban sepsis un 63% mas rapido y tenian un 71% de mayor riesgo de desarrollar sepsis que sus contrapartes no expuestas (RT = 0.37, 95% CI 0.14–1.03; RH = 1.71, 95% CI 1.00–2.94). Los neonatos de madres con corioamnionitis desarrollaban sepsis un 74% mas rapido y tenian un riesgo un 111% mayor de desarrollar sepsis (RT = 0.26, 95% CI 0.07–0.94; RH = 2.11, 95% CI 1.06–4.21). Conclusiones Los neonatos nacidos en Dhaka de madres con colonizacion o corioamnionitis desarrollaron sepsis mas rapido y tenian un mayor riesgo de desarrollar sepsis.

The Aetiology of Neonatal Infection in South Asia (ANISA) study aims to determine the etiology of neonatal infections in 5 population-based sites in Bangladesh, India and Pakistan.The main laboratory challenges in ANISA were selection and consistent implementation of laboratory methods at participating sites with varied infrastructure. The other specific challenges included (1) specimen collection and transport to designated study laboratories and timely processing in rural settings; (2) minimal or nonexistent laboratory facilities at the field sites; (3) obtaining sufficient volumes of blood from enrolled infants aged 0-59 days and (4) caregivers' concerns about collection of clinical specimens from young infants. An additional challenge was selecting an appropriate molecular platform from multiple available options, all with limited field validation, for use in determining infection in young infants.This article describes how the challenges of specimen collection, transport and processing and implementation of laboratory methods have been addressed in the ANISA study. It also describes the measures taken to improve detection of microorganisms causing young infant infections by enhancing the sensitivity of existing laboratory methods for pathogen detection.

The M17 aminopeptidase from the carcinogenic gastric bacterium Helicobacter pylori (HpM17AP) is an important housekeeping enzyme involved in catabolism of endogenous and exogenous peptides. It is implicated in H. pylori defence against the human innate immune response and in the mechanism of metronidazole resistance. Bestatin inhibits HpM17AP and suppresses H. pylori growth. To address the structural basis of catalysis and inhibition of this enzyme, we have established its specificity towards the N-terminal amino acid of peptide substrates and determined the crystal structures of HpM17AP and its complex with bestatin. The position of the D-phenylalanine moiety of the inhibitor with respect to the active-site metal ions, bicarbonate ion and with respect to other M17 aminopeptidases suggested that this residue binds to the S1 subsite of HpM17AP. In contrast to most characterized M17 aminopeptidases, HpM17AP displays preference for L-Arg over L-Leu residues in peptide substrates. Compared to very similar homologues from other bacteria, a distinguishing feature of HpM17AP is a hydrophilic pocket at the end of the S1 subsite that is likely to accommodate the charged head group of the L-Arg residue of the substrate. The pocket is flanked by a sodium ion (not present in M17 aminopeptidases that show preference for L-Leu) and its coordinating water molecules. In addition, the structure suggests that variable loops at the entrance to, and in the middle of, the substrate-binding channel are important determinants of substrate specificity of M17 aminopeptidases.

Due to their lack of toxicity to mammalian cells and good serum stability, proline-rich antimicrobial peptides (PR-AMPs) have been proposed as promising candidates for the treatment of infections caused by antimicrobial-resistant bacterial pathogens. It has been hypothesized that these peptides act on multiple targets within bacterial cells, and therefore the likelihood of the emergence of resistance was considered to be low. Here, we show that spontaneous Escherichia coli mutants resistant to pyrrhocoricin arise at a frequency of approximately 6 × 10 −7 . Multiple independently derived mutants all contained a deletion in a nonessential gene that encodes the putative peptide uptake permease SbmA. Sensitivity could be restored to the mutants by complementation with an intact copy of the sbmA gene. These findings question the viability of the development of insect PR-AMPs as antimicrobials.

Helicobacter pylori infection of the stomach can lead to severe gastroduodenal diseases such as gastritis, peptic ulcers and gastric cancers. Periplasmic H. pylori α-carbonic anhydrase (HpαCA) is essential for the acclimatization of the bacterium to the acidity of the stomach. Through the action of urease and carbonic anhydrases, the H. pylori periplasmic pH is maintained at around 6 in an environment with a pH as low as 2, which in turn facilitates the maintenance of a cytoplasmic pH close to neutral, allowing growth in the gastric niche. Crystals of HpαCA in complex with the inhibitor acetazolamide have been grown by the hanging-drop vapour-diffusion method using polyethylene glycol as a precipitating agent. The crystals have the symmetry of space group P2(1)2(1)2(1), with unit-cell parameters a = 37.0, b = 82.4, c = 150.8 Å. An X-ray diffraction data set was collected from a single crystal to 1.7 Å resolution. Calculation of the self-rotation function using this data and molecular replacement showed that the asymmetric unit contains an HpαCA dimer.

Objectives Some Asian countries have been reluctant to adopt Haemophilus influenzae type b (Hib) conjugate vaccination because of uncertainty over disease burden. We assessed the impact of introduction of Hib conjugate vaccine into the Expanded Program on Immunization in Bangladesh on purulent and laboratory-confirmed H influenzae meningitis. Study design Within a well-defined catchment area around 2 surveillance hospitals in Dhaka, Bangladesh, we compared the incidence of Hib meningitis confirmed by culture, latex agglutination, and polymerase chain reaction assay among infants 1 year before and 1 year after introduction of Hib conjugate vaccine. We adjusted the incidence rate for the proportion of children who sought care at the surveillance hospitals. Results Among infants, the incidence of confirmed Hib meningitis decreased from 92-16 cases per 100 000 within 1 year of vaccine introduction (vaccine preventable incidence = 76; 95% CI 18, 135 per 100 000). The incidence of purulent meningitis decreased from 1659-1159 per 100 000 (vaccine preventable incidence = 500; 95% CI: 203, 799 per 100 000). During the same time period, there was no significant difference in the incidence of meningitis due to Streptococcus pneumoniae. Conclusions Introduction of conjugate Hib conjugate vaccine into Bangladesh Expanded Program on Immunization markedly reduced the burden of Hib and purulent meningitis.

One hundred fifty-two nonculturable lyophilized carriage pneumococcal isolates from a vaccine trial were subjected to PCR for serotyping, and 149 (98%) were successfully classified as vaccine or nonvaccine types, which were similar to viable isolates. The methodology will be useful for analysis of this and other studies where stored pneumococcal isolates fail to grow.