The hpx genetic system for hypoxanthine assimilation as a nitrogen source in Klebsiella pneumoniae: gene organization and transcriptional regulation.

Growth experiments showed that adenine and hypoxanthine can be used as nitrogen sources by several strains of K. pneumoniae under aerobic conditions. The assimilation of all nitrogens from these purines indicates that the catabolic pathway is complete and proceeds past allantoin. Here we identify the genetic system responsible for the oxidation of hypoxanthine to allantoin in K. pneumoniae. The hpx cluster consists of seven genes, for which an organization in four transcriptional units, hpxDE, hpxR, hpxO and hpxPQT, is proposed. The proteins involved in the oxidation of hypoxanthine (HpxDE) or uric acid (HpxO) did not display any similarity to other reported enzymes known to catalyze these reactions, but instead are similar to oxygenases acting on aromatic compounds. Expression of the hpx system is activated by nitrogen limitation and by the presence of specific substrates, with hpxDE and hpxPQT controlled by both signals. Nitrogen control of hpxPQT transcription, which depends on 54, is mediated by the Ntr system. In contrast, neither NtrC nor NAC is involved in the nitrogen control of hpxDE, which is dependent on 70 for transcription. Activation of these operons by the specific substrates is also mediated by different effectors and regulatory proteins. Induction of hpxPQT requires uric acid formation, whereas expression of hpxDE is induced by the presence of hypoxanthine through the regulatory protein HpxR. This LysR-type regulator binds to a TCTGC-N4-GCAAA site in the intergenic hpxD-hpxR region. When bound to this site for hpxDE activation, HpxR negatively controls its own transcription.

Waddlia chondrophila and Chlamydia trachomatis antibodies in screening infertile women for tubal pathology.

Since Waddlia chondrophila is closely related to Chlamydia trachomatis, we hypothesise that W. chondrophila may also be associated with tubal factor infertility (TFI) in women, a major complication of chronic C. trachomatis infection. Five hundred twenty serum samples were tested for anti-Waddlia antibodies by ELISA. Among the 520 investigated women, a total number of 142 (27.3%) has had laparoscopic diagnosis performed, and were either classified TFI positive or negative. Presence of high titres of W. chondrophila antibodies was linked to TFI (p < 0.0001; OR: 7.5; 95% CI: 3.3-17). Moreover, antibody positivity to both W. chondrophila and C. trachomatis-MOMP was strongly associated with TFI (p < 0.0001; OR: 21; 95% CI: 3.8-12E1). This association was much stronger than the statistical association of C. trachomatis-MOMP antibodies only (p < 0.0001; OR: 7.1; 95% CI: 3.7-14), suggesting that co-infection with W. chondrophila and C. trachomatis may lead to more severe reproductive sequelae and immune responses than single infection with either Chlamydiales members.

Improving the molecular diagnosis of Chlamydia psittaci and Chlamydia abortus infection with a species-specific duplex real-time PCR.

Chlamydia psittaci and Chlamydia abortus are closely related intracellular bacteria exhibiting different tissue tropism that may cause severe but distinct infection in humans. C. psittaci causes psittacosis, a respiratory zoonotic infection transmitted by birds. C. abortus is an abortigenic agent in small ruminants, which can also colonize the human placenta and lead to foetal death and miscarriage. Infections caused by C. psittaci and C. abortus are underestimated mainly due to diagnosis difficulties resulting from their strict intracellular growth. We developed a duplex real-time PCR to detect and distinguish these two bacteria in clinical samples. The first PCR (PCR1) targeted a sequence of the 16S-23S rRNA operon allowing the detection of both C. psittaci and C. abortus. The second PCR (PCR2) targeted the coding DNA sequence CPSIT_0607 unique to C. psittaci. The two PCRs showed 100 % detection for ≥ 10 DNA copies per reaction (1000 copies ml- 1). Using a set of 120 samples, including bacterial reference strains, clinical specimens and infected cell culture material, we monitored 100 % sensitivity and 100 % specificity for the detection of C. psittaci and C. abortus for PCR1. When PCR1 was positive, PCR2 could discriminate C. psittaci from C. abortus with a positive predictive value of 100 % and a negative predictive value of 88 %. In conclusion, this new duplex PCR represents a low-cost and time-saving method with high-throughput potential, expected to improve the routine diagnosis of psittacosis and pregnancy complication in large-scale screening programs and also during outbreaks.

Is Switzerland doing enough regarding the prevention of syphilis, gonorrhea and chlamydia infections ?

Sexually transmitted infections are a major problem for medicine and for public health services worldwide. More than 30 sexually transmittable pathogenic micro-organisms are known, including bacteria, viruses, fungi, protozoa and ectoparasites. According to estimates from the World Health Organisation more than 333 million of bacterial sexually transmitted infections occur worldwide per year. Sexually transmitted infections, by their nature, affect individuals, within partnerships and larger sexual networks, and in turn populations. This report focuses on three bacterial sexually transmitted infections in Switzerland that are Chlamydia trachomatis, Neisseria gonorrhea and Treponema pallidum (syphilis) in Switzerland. The prevalence of these infections has been increasing alarmingly for a decade. All three infections can be asymptomatic and their diagnosis and treatment can therefore occur too late or worse not at all, even though treatments are available. This is an important problem as untreated sexually transmitted infections may cause complications such as ascending infections, infertility, ectopic pregnancies and serious long-term neurological sequels. The consequences of these infections should not be underestimated. They constitute a significant public health burden as well as serious financial burden. The increases in chlamydia, syphilis and gonorrhea infections have also been observed in many European countries. Countries, where rising numbers of sexually transmitted infections have been observed, have reacted in different ways. Some have developed clinical guidelines or implemented screening programs, while others are still in their observational phase. The aim of this mémoire is to assess whether Switzerland is doing enough regarding the prevention of chlamydial, syphilis and gonorrheal infections. After first describing the infections, surveillance systems of sexually transmitted infections are assessed, then the epidemiological trends of these three infections are described, and finally the prevention measures implemented in Switzerland to respond to the increasing number of infections are described. The reaction of the United Kingdom to the same problem is reported for comparison. [Author, p. 7]

Importance of amoebae as a tool to isolate amoeba-resisting microorganisms and for their ecology and evolution: the Chlamydia paradigm.

Free-living amoebae are distributed worldwide and are frequently in contact with humans and animals. As cysts, they can survive in very harsh conditions and resist biocides and most disinfection procedures. Several microorganisms, called amoeba-resisting microorganisms (ARMs), have evolved to survive and multiply within these protozoa. Among them are many important pathogens, such as Legionella and Mycobacteria, and also several newly discovered Chlamydia-related bacteria, such as Parachlamydia acanthamoebae, Estrella lausannensis, Simkania negevensis or Waddlia chondrophila whose pathogenic role towards human or animal is strongly suspected. Amoebae represent an evolutionary crib for their resistant microorganisms since they can exchange genetic material with other ARMs and develop virulence traits that will be further used to infect other professional phagocytes. Moreover, amoebae constitute an ideal tool to isolate strict intracellular microorganisms from complex microbiota, since they will feed on other fast-growing bacteria, such as coliforms potentially present in the investigated samples. The paradigm that ARMs are likely resistant to macrophages, another phagocytic cell, and that they are likely virulent towards humans and animals is only partially true. Indeed, we provide examples of the Chlamydiales order that challenge this assumption and suggest that the ability to multiply in protozoa does not strictly correlate with pathogenicity and that we should rather use the ability to replicate in multiple and diverse eukaryotic cells as an indirect marker of virulence towards mammals. Thus, cell-culture-based microbial culturomics should be used in the future to try to discover new pathogenic bacterial species.

Permissivity of fish cell lines to three Chlamydia-related bacteria: Waddlia chondrophila, Estrella lausannensis and Parachlamydia acanthamoebae.

Epitheliocystis is an infectious disease affecting gills and skin of various freshwater and marine fishes, associated with high mortality and reduced growth of survivors. Candidatus Piscichlamydia salmonis and Clavochlamydia salmonicola have recently been identified as aetiological agents of epitheliocystis in Atlantic Salmon. In addition, several other members of the Chlamydiales order have been identified in other fish species. To clarify the pathogenicity of Chlamydia-like organisms towards fishes, we investigated the permissivity of two fish cell lines, EPC-175 (Fathead Minnow) and RTG-2 (rainbow trout) to three Chlamydia-related bacteria: Waddlia chondrophila, Parachlamydia acanthamoebae and Estrella lausannensis. Quantitative PCR and immunofluorescence demonstrated that W. chondrophila and, to a lesser extent, E. lausannensis were able to replicate in the two cell lines tested. Waddlia chondrophila multiplied rapidly in its host cell and a strong cytopathic effect was observed. During E. lausannensis infection, we observed a limited replication of the bacteria not followed by host cell lysis. Very limited replication of P. acanthamoebae was observed in both cell lines tested. Given its high infectivity and cytopathic effect towards fish cell lines, W. chondrophila represents the most interesting Chlamydia-related bacteria to be used to develop an in vivo model of epitheliocystis disease in fishes.

Chlamydiaceae and Chlamydia-like organisms in the koala (Phascolarctos cinereus)--organ distribution and histopathological findings.

Chlamydial infections in koalas can cause life-threatening diseases leading to blindness and sterility. However, little is known about the systemic spread of chlamydiae in the inner organs of the koala, and data concerning related pathological organ lesions are limited. The aim of this study was to perform a thorough investigation of organs from 23 koalas and to correlate their histopathological lesions to molecular chlamydial detection. To reach this goal, 246 formalin-fixed and paraffin embedded organ samples from 23 koalas were investigated by histopathology, Chlamydiaceae real-time PCR and immunohistochemistry, ArrayTube Microarray for Chlamydiaceae species identification as well as Chlamydiales real-time PCR and sequencing. By PCR, two koalas were positive for Chlamydia pecorum whereas immunohistochemical labelling for Chlamydiaceae was detected in 10 tissues out of nine koalas. The majority of these (n=6) had positive labelling in the urogenital tract related to histopathological lesions such as cystitis, endometritis, pyelonephritis and prostatitis. Somehow unexpected was the positive labelling in the gastrointestinal tract including the cloaca as well as in lung and spleen indicating systemic spread of infection. Uncultured Chlamydiales were detected in several organs of seven koalas by PCR, and four of these suffered from plasmacytic enteritis of unknown aetiology. Whether the finding of Chlamydia-like organisms in the gastrointestinal tract is linked to plasmacytic enteritis is unclear and remains speculative. However, as recently shown in a mouse model, the gastrointestinal tract might play a role being the site for persistent chlamydial infections and being a source for reinfection of the genital tract.

Parachlamydia spp. and related Chlamydia-like organisms and bovine abortion.

Chlamydophila abortus and Waddlia chondrophila cause abortion in ruminants. We investigated the role of Parachlamydia acanthamoebae in bovine abortion. Results of immunohistochemical analyses were positive in 30 (70%) of 43 placentas from which Chlamydia-like DNA was amplified, which supports the role of Parachlamydia spp. in bovine abortion.

Emerging, non-PCV13 serotypes 11A and 35B of Streptococcus pneumoniae show high potential for biofilm formation in vitro

Background: Since the use of pneumococcal conjugate vaccines PCV7 and PCV13 in children became widespread, invasive pneumococcal disease (IPD) has dramatically decreased. Nevertheless, there has been a rise in incidence of Streptococcus pneumoniae non-vaccine serotypes (NVT) colonising the human nasopharynx. Nasopharyngeal colonisation, an essential step in the development of S. pneumoniae-induced IPD, is associated with biofilm formation. Although the capsule is the main pneumococcal virulence factor, the formation of pneumococcal biofilms might, in fact, be limited by the presence of capsular polysaccharide (CPS). Methodology/Principal Findings: We used clinical isolates of 16 emerging, non-PCV13 serotypes as well as isogenic transformants of the same serotypes. The biofilm formation capacity of isogenic transformants expressing CPSs from NVT was evaluated in vitro to ascertain whether this trait can be used to predict the emergence of NVT. Fourteen out of 16 NVT analysed were not good biofilm formers, presumably because of the presence of CPS. In contrast, serotypes 11A and 35B formed >45% of the biofilm produced by the non-encapsulated M11 strain. Conclusions/Significance This study suggest that emerging, NVT serotypes 11A and 35B deserve a close surveillance.

Infecção por Chlamydia trachomatis e Neisseria gonorrhoeae em mulheres atendidas em serviço de planejamento familiar

OBJETIVO: estudar a prevalência de infecções por Chlamydia trachomatis (CT) e Neisseria gonorrhoeae (NG) entre mulheres adolescentes e jovens em ambulatório de planejamento familiar. MÉTODOS: um total de 230 mulheres com idade menor ou igual a 24 anos e antecedente de até quatro parceiros sexuais foram acompanhadas por até 48 meses, com coletas de urina para pesquisa de CT e NG pelo método da reação em cadeia da polimerase nos meses 1, 12, 24, 36 e 48. As variáveis estudadas foram faixa etária, escolaridade, estado marital, número de gestações, abortos e filhos vivos, idade de início da vida sexual, uso anterior e atual de condom, uso anterior de dispositivo intrauterino, número de parceiros nos últimos seis meses e tempo de seguimento. Realizou-se análise bivariada das variáveis segundo os testes positivos para CT e NG e análise múltipla por regressão logística. RESULTADOS: a frequência de infecções por CT foi de 13,5% e por NG de 3%, duas mulheres apresentaram ambos os testes positivos. O antecedente de uso de dispositivo intrauterino foi associado aos testes positivos para NG. CONCLUSÕES: as prevalências de infecção por CT e NG foram altas na faixa etária estudada e o rastreamento de mulheres jovens deve ser considerado em nossos serviços para controle da disseminação e prevenção de sequelas das doenças sexualmente transmissíveis.

Prevalência da infecção por Chlamydia Trachomatis e Neisseria Gonorrhoea em mulheres jovens sexualmente ativas em uma cidade do Sul do Brasil

OBJETIVO: conhecer a prevalência da clamídia e da gonorreia numa amostra de mulheres da cidade de Curitiba. MÉTODOS: estudo transversal com mulheres sexualmente ativas entre 16 e 23 anos, útero intacto, não gestantes, com até quatro parceiros sexuais, sem evidência de cervicite purulenta ou febre e que foram submetidas a exame pélvico e avaliadas pelo método PCR em amostra de urina para clamídia e gonorreia. Os critérios de exclusão foram: vacinação para o HPV, história de vacinação nos últimos 21 dias, citologia oncótica anterior anormal, história de verrugas genitais, esplenectomia, distúrbios imunológicos e uso de imunossupressores. Foi aplicada entrevista contendo dados sociodemográficos, gineco-obstétricos e de comportamento de risco para doença sexualmente transmissível. Para a análise estatística, utilizou-se o teste do c2 ou o teste exato de Fisher para avaliar a associação entre as variáveis. RESULTADOS: a prevalência da infecção por clamídia e gonorreia no grupo estudado foi de 10,7 e 1,5%, respectivamente, sendo a taxa de coinfecção de 0,9%. Não foi encontrada correlação entre as faixas de idade das voluntárias, a idade de início da atividade sexual, o número de parceiros sexuais e o número de novos parceiros sexuais nos últimos seis meses, com a presença de clamídia ou gonorreia. As mulheres que apresentavam corrimento ou ectrópio tiveram uma prevalência de infecção por clamídia duas vezes mais alta do que aquelas que não apresentavam esses sinais. CONCLUSÕES: os resultados foram similares aos estudos nacionais, utilizando PCR em amostra de urina para detecção de clamídia e gonorreia, com amostras de mulheres não gestantes nas mesmas faixas de idade e com os mesmos antecedentes. Por terem sido excluídas as voluntárias com mais de quatro parceiros sexuais e aquelas que apresentavam endocervicite purulenta, acredita-se que a prevalência da infecção pela clamídia e gonorreia poderia ter sido maior na população estudada.