Characterisation of extended-spectrum b-lactamases among Klebsiella pneumoniae isolates causing bacteraemia and urinary tract infection in Mozambique

The aim of this study was to determine the prevalence of extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae isolated from urinary tract and bloodstream infections in a rural hospital in Manhiça, Mozambique. ESBLs were investigated among ceftriaxone-non-susceptible K. pneumoniae clinical isolates recovered between 2004 and 2009. Characterisation of blaCTX-M, blaSHV, blaOXA and blaTEM genes was performed by PCR and sequencing. Epidemiological relationships were established by phylogenetic analysis, repetitive extragenic palindromic PCR (REP-PCR), pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST), whilst plasmid transferability was evaluated by conjugation. In addition, the presence of class 1 and 2 integrons was studied. A total of 19 K. pneumoniae were analysed. The blaCTX-M-15 gene was found in all strains. Other ESBL genes were found concomitantly, including blaSHV-5, blaSHV-2, blaSHV-2A, blaSHV-12 and blaSHV-38. In addition, other β-lactamases such as blaTEM-1 and blaOXA-30 were also detected. REP-PCR identified 15 different epidemiological profiles. MLST analysis also showed great variability of sequence types. The blaCTX-M-15 gene showed a high transfer capacity. The presence of class 1 integrons was high. High levels of multidrug resistance were also found. In conclusion, these data show the dominance of the CTX-M-type ESBL, particularly CTX-M-15, supporting its worldwide dissemination, including in areas with limited access to third-generation cephalosporins. This finding is a matter of concern for clinical management as third-generation cephalosporins are an alternative for treating severe cases of multidrug-resistant infections in this community

Escherichia coli ST131 clones harbouring AggR and AAF/V fimbriae causing bacteremia in Mozambican children: Emergence of new variant of fimH27subclone

Multidrug-resistant Escherichia coli ST131 fimH30 responsible for extra-intestinal pathogenic (ExPEC) infections is globally distributed. However, the occurrence of a subclone fimH27 of ST131 harboring both ExPEC and enteroaggregative E. coli (EAEC) related genes and belonging to commonly reported O25:H4 and other serotypes causing bacteremia in African children remain unknown. We characterized 325 E. coli isolates causing bacteremia in Mozambican children between 2001 and 2014 by conventional multiplex polymerase chain reaction and whole genome sequencing. Incidence rate of EAEC bacteremia was calculated among cases from the demographic surveillance study area. Approximately 17.5% (57/325) of isolates were EAEC, yielding an incidence rate of 45.3 episodes/105 children-years-at-risk among infants; and 44 of isolates were sequenced. 72.7% (32/44) of sequenced strains contained simultaneously genes associated with ExPEC (iutA, fyuA and traT); 88.6% (39/44) harbored the aggregative adherence fimbriae type V variant (AAF/V). Sequence type ST-131 accounted for 84.1% (37/44), predominantly belonging to serotype O25:H4 (59% of the 37); 95.6% (35/44) harbored fimH27. Approximately 15% (6/41) of the children died, and five of the six yielded ST131 strains (83.3%) mostly (60%; 3/5) due to serotypes other than O25:H4. We report the emergence of a new subclone of ST-131 E. coli strains belonging to O25:H4 and other serotypes harboring both ExPEC and EAEC virulence genes, including agg5A, associated with poor outcome in bacteremic Mozambican children, suggesting the need for prompt recognition for appropriate management

Molecular characterization of Klebsiella pneumoniae β-lactamases from patients admitted at the University Hospital of Coimbra, Portugal

Dissertação de Mestrado em Biologia Celular e Molecular, apresentada ao Departamento de Ciências da Vida da Faculdade de Ciências e Tecnologia da Universidade de Coimbra.A emergência e disseminação de bactérias resistentes aos antibióticos é um problema global e representa uma grande ameaça para a saúde dos pacientes. Com importância especial é a disseminação de β-lactamases com capacidade de inativar quase todos os antibióticos β- lactámicos contribuindo para limitação das opções de tratamento. Portanto uma rápida deteção e caracterização dos determinantes de resistência constituem uma ferramenta importante para o tratamento dos pacientes e melhoramento das medidas de controlo de infeções. O estudo teve como objetivo caracterizar geneticamente isolados de Klebsiella pneumoniae resistentes ao ertapenem de pacientes admitidos nos Hospitais da Universidade de Coimbra, de Janeiro a Dezembro de 2013. Os isolados foram testados a sua suscetibilidade aos antibióticos usando o método de disco difusão. A PCR convencional foi usada para deteção de genes que codificam as β-lactamases e para a tipagem dos plasmídeos, enquanto a relação clonal dos isolados foi determinada com recurso as técnicas de PFGE e MLST. Os ensaios de conjugação foram feitos para avaliar o potencial de transferência dos genes de resistência entre bactérias. Foram analisados 27 isolados de K. pneumoniae não duplicados e a maioria foi de urina 48.1% (13/27) e sangue 25.9% (7/27). Foi observada uma alta taxa de resistência aos antibióticos associada a produção de β-lactamases. As beta-lactamases de espectro estendido (ESBL) foram o principal mecanismo de resistência encontrado, com TEM (77.7%), o mais prevalente seguido de CTX-M (70.3%). A carbapenemase-KPC foi detetada em 66.6% dos isolados. Este é o primeiro reporte de KPC-2 em isolados nosocomiais de K. pneumoniae nos hospitais da Universidade de Coimbra, e em Portugal. A KPC estava associada a um plasmídeo conjugativo do grupo F oque sustenta a sua disseminação e capacidade de transmissão para outras bactérias. Análises de PFGE mostraram um número limitado de clones representados por três diferentes perfis de MLST, ST11, ST15 e ST348, oque sugere a presença de clones específicos no hospital. Uma contínua vigilância de resistências aos antibióticos e uso racional dos antibióticos é necessária para melhorar o tratamento dos pacientes e para o controlo das infeções.The emergence and spread of antibiotic resistant bacteria is a global concern and represents a major threat for patient care. Of particular importance is the dissemination of β-lactamases with ability to inactivate almost all β-lactam antibiotics, leading to the limitation of treatment options. Therefore, rapid detection and characterization of resistant determinants is an important tool for patient management and improvement of infection control measures. The present study aimed to characterize genetically a collection of isolates of Klebsiella pneumoniae ertapenem resistant recovered from patients at the University Hospital of Coimbra from January-December, 2013. All isolates were tested for antimicrobial susceptibility by disk diffusion method. Conventional PCR was used to detect β-lactamases genes and for plasmid typing while clonal relatedness of the isolates was carried out by PFGE and MLST analysis. Finally, plasmid conjugation assays were conducted to verify the potential of transfer of antibiotic resistance determinants among bacteria. A total number of 27 non-duplicated K. pneumoniae isolates have been analyzed and the majority was recovered from urine 48.1% (13/27) and blood 25.9% (7/27). A high prevalence of antibiotic resistance associated with production of β-lactamases, including to carbapenems, was observed. Extended spectrum β-lactamases (ESBLs) was the more frequent resistance mechanism found, with TEM-type β-lactamase (77.7%) being the most common found followed by CTX-M-type (70.3%). KPC- carbapenemase was detected in 66.6% of the isolates. This is the first report of KPC-2 in nosocomial isolates of K. pneumoniae in the referral University Hospital of Coimbra and in Portugal. KPC was associated with conjugative IncF plasmid type which supports their dissemination and potential spreading to other bacterial. PFGE analysis showed a limited number of clones represented by three MLST profiles, ST11, ST15 and ST348, which suggest the presence of specific clones in the hospital. Continuous surveillance of antibiotic resistance and rational use of antibiotics is needed to improve patient management and infection control measures

Characterisation of extended-spectrum b-lactamases among Klebsiella pneumoniae isolates causing bacteraemia and urinary tract infection in Mozambique

The aim of this study was to determine the prevalence of extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae isolated from urinary tract and bloodstream infections in a rural hospital in Manhiça, Mozambique. ESBLs were investigated among ceftriaxone-non-susceptible K. pneumoniae clinical isolates recovered between 2004 and 2009. Characterisation of blaCTX-M, blaSHV, blaOXA and blaTEM genes was performed by PCR and sequencing. Epidemiological relationships were established by phylogenetic analysis, repetitive extragenic palindromic PCR (REP-PCR), pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST), whilst plasmid transferability was evaluated by conjugation. In addition, the presence of class 1 and 2 integrons was studied. A total of 19 K. pneumoniae were analysed. The blaCTX-M-15 gene was found in all strains. Other ESBL genes were found concomitantly, including blaSHV-5, blaSHV-2, blaSHV-2A, blaSHV-12 and blaSHV-38. In addition, other β-lactamases such as blaTEM-1 and blaOXA-30 were also detected. REP-PCR identified 15 different epidemiological profiles. MLST analysis also showed great variability of sequence types. The blaCTX-M-15 gene showed a high transfer capacity. The presence of class 1 integrons was high. High levels of multidrug resistance were also found. In conclusion, these data show the dominance of the CTX-M-type ESBL, particularly CTX-M-15, supporting its worldwide dissemination, including in areas with limited access to third-generation cephalosporins. This finding is a matter of concern for clinical management as third-generation cephalosporins are an alternative for treating severe cases of multidrug-resistant infections in this community

Genomic analysis of sewage from 101 countries reveals global landscape of antimicrobial resistance

Antimicrobial resistance (AMR) is a major threat to global health. Understanding the emergence, evolution, and transmission of individual antibiotic resistance genes (ARGs) is essential to develop sustainable strategies combatting this threat. Here, we use metagenomic sequencing to analyse ARGs in 757 sewage samples from 243 cities in 101 countries, collected from 2016 to 2019. We find regional patterns in resistomes, and these differ between subsets corresponding to drug classes and are partly driven by taxonomic variation. The genetic environments of 49 common ARGs are highly diverse, with most common ARGs carried by multiple distinct genomic contexts globally and sometimes on plasmids. Analysis of flanking sequence revealed ARG-specific patterns of dispersal limitation and global transmission. Our data furthermore suggest certain geographies are more prone to transmission events and should receive additional attention