Lactic acid bacteria are associated with preservation of foods, including milk, meat and vegetables. Yoghurt is produced by the cooperative action of two starter bacteria / S. thermophilus and L. delbrueckii subsp. bulgaricus. In this study, identification and typing of yoghurt starter bacteria were aimed. Traditional home made yoghurts were collected from different areas of Turkey, identification of those isolates at species and subspecies level and typing at strain level were achieved using PCR based methods. In our study, identification of yogurt starter bacteria was studied using species specific primers and ARDRA. These methods were inefficient in identification of yoghurt starter bacteria, at species and subspecies level. Consequently, a reliable and quick method for accurate identification of yoghurt starter bacteria was developed. The new method focuses on amplification of methionine biosynthesis genes, for selective identification of yoghurt starter bacteria together with some cheese starters. Further discrimination by ARDRA enabled differentiation of yoghurt starter bacteria from cheese starters. Confirmation of the proposed method has been accomplished by partial sequencing of the 16S rRNA gene. After correct identification of starter bacteria had been achieved, the strains were typed at strain level using RAPD-PCR and MLST. RAPD-PCR with primer 1254 resulted better fingerprints, compared to primer M13 at strain level. Comparisons of the two typing methods showed that RAPD-PCR revealed strain diversity better than MLST, however MLST was a more robust and reliable method and resulted in clustering of the strains depending on the isolation source.

Staphylococcus aureus has long been recognized as a leading cause of nosocomial infection. However, several recent publications have demonstrated this pathogen as the cause of community-acquired severe wound infections and necrotizing pneumonia in otherwise healthy individuals. These highly virulent endemic clones have been reported in several locations in the United States and Canada. The rapid spread of the organism, the ability of certain clones to cause serious infection, and the antibiotic resistance of the endemic clones, illustrates the importance of infection control measures. In this study we examined three S. aureus typing techniques; pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and Staphylococcal protein A (spa) sequencing for subspeciation of community-acquired methicillin-resistant S. aureus (CA-MRSA). It is hypothesized that PFGE will result in a higher level of discrimination among the strains, while MLST and spa typing will result in highly portable data that lacks the discriminatory power of PFGE. Thirty CA-MRSA isolates that were obtained from Florida and Washington State were characterized by molecular typing methods. Whole genome restriction analysis was performed by PFGE using the SmaI enzyme. Sequence-based typing analyses, MLST and spa typing, were performed by polymerase chain reaction (PCR) followed by sequencing. PFGE data was analyzed using the BioNumerics® software package and sequence-based data was analyzed using DNAstar®. MLST Alleles were assigned using the online MLST database (www.mlst.net) and spa types were assigned using the Ridom SpaServer (www.ridom.de/spaserver). Molecular characterization of the 30 isolates resulted in 21 pulsotypes, four MLST sequence types (STs), and six spa types. Combining data from both MLST and spa typing resulted in only seven strain categories, many of which grouped isolates that are not epidemiologically linked. These data demonstrate that techniques such as MLST and spa typing are not well suited for tracking isolates with limited evolutionary diversity such as the CA-MRSA epidemic clones.

Staphylococcus epidermidis has emerged in recent years as an important nosocomial pathogen, especially in infections associated with implanted foreign body materials (e.g., prosthetic joints and heart valves) and in individuals with a compromised immune system (e.g., cancer patients and neonates). Although rare, implant infections are long lasting and cause severe suffering for the patient that includes pain and disability and even increased mortality. One aim of the present thesis was to develop and evaluate a genetic method for species identification and simultaneous detection of rifampicin resistance in staphylococci. A second aim was to examine S. epidermidis isolated from prosthetic joint infections (PJIs) and from wrists and nares of healthy individuals regarding their antibiotic susceptibility, biofilm production, virulence factors, and epidemiology. Comparison with phenotypic diagnostics revealed that 8 (16%) of 49 isolates differed in their species identification in favour of the genetic method. In addition, mutations associated with rifampicin resistance, including two not previously reported, were possible to detect in all isolates resistant to rifampicin. Antibiotic susceptibility testing of 61 PJI isolates showed multi-drug resistance in 91%. Furthermore, the results of the synergy testing revealed that no antibiotic combination was significantly better than the others. Hence, the effects that were possible to detect were isolate dependent. To find a method for discriminating between invasive (n=61) and commensal (n=24) isolates of S. epidermidis genotypic and phenotypic characterisations of biofilm production (including the ica and aap genes), antibiotic susceptibility, virulence-related genes (such as agr and ACME) and epidemiology were performed (using multilocus sequence typing [MLST], typing of the staphylococcal chromosome cassette mec [SCCmec] and PhenePlate). Significant differences were found in antibiotic susceptibility, i.e. there was more resistance among invasive isolates. MLST sequence types (ST) ST2 and ST215 dominated the invasive isolates.

During the evolution some groups of organisms have become coevolutionary associated with other groups, as is the case of host symbiont systems. To explore coevolutionary history of hosts and their associated symbionts, phylogenetic reconstruction of symbionts and phylogenetic reconstruction of hosts are usually compared. Coevolution is described by coevolutionary events (cospeciation, host switch, duplication, failure to diverge events and linage sorting events). The aim of this work was to test the suitability of MLST method for the complex of entomopathogenic nematodes from the genus Steinernema (with detailed analysis of Steinernema feltiae) and their symbiotic bacteria Xenorhabdus bovienii and subsequently use cophylogenetic comparative analysis to determine their level of cospeciation.

Orientador: Maria de Lourdes Ribeiro de Souza da Cunha / Resumo: Indivíduos colonizados podem contribuir para disseminação de Staphylococcus aureus, que se destaca por sua patogenicidade e alta frequência, capaz de produzir doenças tanto em indivíduos sadios quanto em imunocomprometidos, sendo esse risco aumentado na imunodeficiência humana (HIV/aids). Esse estudo teve como objetivos determinar a relação clonal de MRSA (Staphylococcus aureus Resistente à Meticilina) e MSSA (Staphylococcus aureus Sensível à Meticilina) entre as cepas isoladas de pessoas vivendo com HIV/aids (PVHA) e seus contactantes domiciliares, e os fatores de virulência que podem contribuir para o carreamento desses micro-organismos. S. aureus foram isolados da nasofaringe e orofaringe de 368 PVHA do Serviço de Ambulatórios Especializados de Infectologia “Domingos Alves Meira” (SAE/DAM) do complexo da Faculdade de Medicina de Botucatu (FMB) e Fundação para o Desenvolvimento Médico Hospitalar (FAMESP), sendo 112 residentes em Botucatu e 256 provenientes de cidades da região. A técnica de PCR (Reação em Cadeia da Polimerase) foi utilizada para detecção do gene mecA, genes das enterotoxinas (sea, seb, e sec-1), esfoliatinas A e B (eta e etb), toxina 1 da síndrome do choque tóxico (tst), leucocidina Panton–Valentine (lukS-PV e lukF-PV), hemolisinas alfa e delta (hla and hld) e biofilme (icaA e icaD) e para tipagem do SCCmec. Para a tipagem molecular foi utilizado a técnica de Pulsed Field Gel Electrophoresis (PFGE), Multilocus Sequence Typing (MLST) e spa Typing. Os pacient... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Colonized individuals may contribute to the dissemination of Staphylococcus aureus, which stands out for its pathogenicity and high frequency, capable of producing diseases in both healthy and immunocompromised individuals, with an increased risk of human immunodeficiency (HIV/AIDS). This study aimed to determine the clonal relationship between MRSA (Methicillin Resistant Staphylococcus aureus) and MSSA (Methicillin-Sensitive Staphylococcus aureus) between strains isolated from people living with HIV/aids (PLHA) and their home contacts, and virulence factors which may contribute to the transport of these microorganisms. S. aureus were isolated from the nasopharynx and oropharynx of 368 PLHA from the Specialized Outpatient Services “Domingos Alves Meira” (SAE/DAM) of the Botucatu Medical School (FMB) and Hospital Medical Development Foundation (FAMESP), of which 112 were residents in Botucatu and 256 from cities in the region. The PCR (Polymerase Chain Reaction) technique was used to detect the mecA gene, enterotoxin genes (sea, seb, and sec-1), esfoliatins A and B (eta and etb), toxic shock syndrome toxin 1 (tst), Panton-Valentine leukocidin (lukS-PV and lukF-PV), alpha and delta hemolysins (hla and hld) and biofilm (icaA and icaD) and for typing SCCmec. For molecular typing were used techniques of the Pulsed Field Gel Electrophoresis (PFGE), Multilocus Sequence Typing (MLST) and spa Typing. The patients residing in Botucatu were invited to more two collections with the inclu... (Complete abstract click electronic access below) / Doutor

The increasing number of Extended Spectrum Beta-Lactamase (ESBL) producing Escherichia coli (E. coli) associated with sepsis in China is the reason for designing the current study. During 2014-2016, thirty hospitals representing 10 different provinces in China was involved in collecting E. coli isolates causing blood stream infections. Early treatment with suitable antibiotics have been found to be of lifesaving importance in the case of care for septic patients. Thorough understanding of the pathogens involved is therefore crucial. Using antimicrobial susceptibility testing, PCR and Multi Locus Sequence Typing (MLST), the molecular characteristics of ESBL producing E. coli isolates could be determined. This study can report that the most common ESBL producing genes found were CTX-M-14 (51 isolates, 45,5%), CTX-M-55 (23 isolates, 20,5%) CTX-M-15 (22 isolates, 19,6%). In addition, 2 isolates (1,8%) were found to be SHV-11 positive which is another ESBL producing gene. As a side finding, 5 isolates harbored Metallo-beta-lactamase (MBL) encoding genes such as NDM-5 and NDM-1 which were found to coexist with CTX-M-55 and CTX-M-14 respectively. An MLST analysis resulted in the finding of 25 different and 17 previously unknown (16,2 %) sequence types. The most common sequence types were ST131 (18 isolates, 17,1 %) as reported previously.  No significant differences in antimicrobial susceptibility were identified whether ESBL producing genes such as SHV and CTX-M was present or not. This study indicates that there could be novel resistance mechanisms present among those isolates not encoding the genes of interest. However, this finding requires further research before it can be confirmed.

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Yes / Background: Group B Streptococcus (GBS) is a leading cause of morbidity and mortality in infants in both developed and developing countries. To our knowledge, only a few studies have been reported the clinical features, treatment and outcomes of the GBS disease in China. The severity of neonatal GBS disease in China remains unclear. Population-based surveillance in China is therefore required. Methods: We retrospectively collected data of <3 months old infants with culture-positive GBS in sterile samples from three large urban tertiary hospitals in South China from Jan 2011 to Dec 2014. The GBS isolates and their antibiotic susceptibility were routinely identified in clinical laboratories in participating hospitals. Serotyping and multi-locus sequence typing (MLST) were also conducted for further analysis of the neonatal GBS disease. Results: Total 70 cases of culture-confirmed invasive GBS infection were identified from 127,206 live births born in studying hospitals, giving an overall incidence of 0.55 per 1000 live births (95% confidence interval [CI] 0.44–0.69). They consisted of 49 with early-onset disease (EOD, 0.39 per 1000 live births (95% CI 0.29–0.51)) and 21 with late-onset disease (LOD, 0.17 per 1000 live births (95% CI 0.11–0.25)). The incidence of EOD increased significantly over the studying period. Five infants (4 EOD and 1 LOD) died before discharge giving a mortality rate of 7.1% and five infants (7.1%, 2 EOD and 3 LOD) had neurological sequelae. Within 68 GBS isolates from GBS cases who born in the studying hospitals or elsewhere, serotype III accounted for 77.9%, followed by Ib (14.7%), V (4.4%), and Ia (2.9%). MLST analysis revealed the presence of 13 different sequence types among the 68 GBS isolates and ST-17 was the most frequent sequence type (63.2%). All isolates were susceptible to penicillin, ceftriaxone, vancomycin and linezolid, while 57.4% and 51.5% were resistant to erythromycin and clindamycin, respectively. Conclusions: This study gains the insight into the spectrum of GBS infection in south China which will facilitate the development of the guidance for reasonable antibiotics usage and will provide evidence for the implementation of potential GBS vaccines in the future. / Supported by medical and health science and technology projects of Health and Family Planning Commission of Guangzhou Municipality (grant number 20151A010034) and Guangdong provincial science and technology planning projects (grant number 2014A020212520).