Phenotypic and molecular characterization of extraintestinal pathogenicEscherichia coliand other Gram-negative invasive bacteria in Mozambique

There is limited information is available regarding the population structure of extraintestinal pathogenic Escherichia coli (ExPEC), and other Gram-negative bacteria (GNB) and their susceptibility profile in Africa, although the increasing prevalence of Extended Spectrum β-lactamases (ESBL), Plasmid mediated β-lactamases (AmpC) and Carbapenemase producing bacteria. Characterization of Gram-negative bacteria pathogens (GNBP) is pivotal in selection of empiric antimicrobials, while awaiting bacterial culture results with suspected invasive bacterial and infection preventive measures worldwide. In the majority of low-income countries like Mozambique, despite the increasing of GNB that produces ESBLs, Plasmid mediated β-lactamases (AmpC) and Carbapenemase and their association with high morbidity-mortality, not all antibiotics agents are available to treat these pathogens and most clinical diagnostic laboratories may not attempt to detect these three major groups of enzymes leading to difficulties in the hospital control of resistant microorganisms and antibiotics misuse. The overall purpose of this study was to evaluate the epidemiology and antimicrobial resistance of ExPEC and other GNB associated with invasive infections such as urinary tract infection, intra-abdominal infection, osteomyelitis, soft-tissue infection, pneumonia, sepsis, meningitis and other infections in Mozambique, through phenotypic and molecular approach. From February 2016 to July of 2018, mainly in Hospital Central of Maputo (HCM) and in other four hospitals: Hospital General of Macamo (HGM), Hospital General of José Macamo (HJM) Hospital Provincial of Quelimane (HPQ) and Hospital Central of Quelimane (HCQ) (n=159) samples were collected. Bacterial identification was done using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF). Antibiotic Susceptibility Testing (AST) was done phenotypically by VITEK 2 compact system (bioMérieux) and by genotypic approaches through PCR (n=77) and Whole Genome Sequencing (WGS) (n=82) DNA of Gram-negative bacteria extracted using Wizard® Genomic DNA Purification Kit (Promega). Full genome sequences of the isolates were obtained using WGS technology and were analyzed in silico for molecular characterization. To our best knowledge, this is the first depth study of ExPEC and other GNB based on WGS in Mozambique. Overall, 159 GNB were isolated, of which 81% were from the main Departments of HCM (Pediatric, Medicine, Surgery and Gynecology and Obstetrics) and 19% from other hospitals (HGM and HJM, and HPQ and HCQ), in particular from Pediatric Departments. Of the 128 GNB from HCM, 59% were isolated from blood and 38% and 2% from pus and cerebrospinal fluid (CSF), respectively.The most frequent isolates in HCM were K. pneumoniae (27%), followed by ExPEC and Acinetobacter spp. both with 20%. Other GNB as Pseudomonas spp., P. mirabilis, Enterobacter spp., Salmonella spp., M. morgani, K. oxytoca, K. variicola, and S. maltophilia were less observed (ranging from 8% to 1%). The Acinetobacter spp. included (n=23) A. baumanni, and (n=1) A. complex; Pseudomonas spp. included (n=17) P. aeruginosa, (n=1) P. otitidis and (n=1) P. stutzeri; Enterobacter spp. included (n=4) E. complex and (n=1) E. cloacae; and Salmonella spp. included, (n=2) Salmonella enterica serovar Typhimurium (S. Typhimurium), and (n=1) S. Isangi. From other hospitals, seven GNB from blood samples were isolated, including (n=2) Salmonella spp. (n=8) S. Typhimurium and (n=1) Salmonella enterica serovar Thompson, (4) E. coli and K. pneumoniae, (3) E. cloacae, (2) P. aeruginosa, (n=2) A. baumanni and A. complex, (n=1) S. complex, S. marcescens and P. septica only in Pediatric departments. Among ExPEC and other GNB, the resistance to the commonly antibiotics, including penicillins, cephalosporins, trimethoprim-sulfamethoxazole, gentamicin and ciprofloxacin used in Mozambique is high and being associated with different mechanisms, including narrow β-lactamases (TEM-1B, OXA-1), ESBL (CTX-M-9, 15, -27, -88), AmpC (CMY-2 and DHA-13) and carbapenemases (NDM-5, OXAs, NDM and VIM). These mechanisms were found associated with others depending on the species, namely: chromosomal mutations Fluoroquinolone mutations in gyrA, parC and parE, acrR, ompK36, ompK37 and ramR; efflux pumps oqxA and oqxB, aminoglycosides and fluoroquinolones genes [aac(6')-Iaa, aac(6')Ib-cr, aac(6')-Ian, aac(6')-IIC, aac(3)-Ia, aac(3)-IIa, aac(3)-IId, aph(3')-Ia, aph(3'')-I, aph(6)-Id and ant(2'')-Ia, 16S rRNA methylases (rmtB)], sulphonamides (sul1 and sul2), phenicols (catA1, catA2, catA3, catB3, catB7, cmlA1 and floR), macrolides [mdf(A), mph(A), mph(E) and msr(E) and ereA), tetracycline [tet(A)/(B), tet(D), tet(J) and tet(39)], fosfomycin (fosA), rifampicin (ARR-2 and ARR-3), plasmid mediated quinolone resistance (qnrB1, qnrB6 and qnrD1), colistin (mcr-9) and dfr genes, mainly belonging to class 1 and 2 integrons (dfrA1, drfrA5, dfrA7, dfrA8, dfrA12, dfrA14, dfrA17, dfrA16, dfrA19, dfrA27) encoding resistance to trimethoprim-sulfamethoxazole.Several GNB including ExPEC, K. pneumoniae, P. mirabilis, K. oxytoca, and E. complex, harbored the ESBL CTX-M-15 downstream of ISEcp1 on IncF and A/C plasmids, that plays an important role in the dissemination of resistance genes among GNB in HCM.The presence of pandemic or/and emerging ExPEC (mainly ST131, ST69, ST410, ST405, ST38), K. pneumoniae, K. oxytoca, E. cloacae ST84, P. mirabilis, A. baumanii and P. aeruginosa carrying CTX-M-15 and ISEcp1 linked to IncF, A/C and Col plasmids represent a high risk for the country due to rapidly dissemination and evolution of diverse multiresistant plasmids and also for treatment failure with antibiotics. Additionally, the detection of the hypervirulent and hypermucoviscous K. pneumoniae ST23 constitutes a worrisome situation due to its ability to generate invasive community-acquired infections. Therefore, prudent use of antibiotics is advocated, and a systematic national surveillance system of antibiotic resistance is urgently need to overcome the dissemination of ESBL, AmpC, and carbapenemases containing GNB in Mozambique. The presence of E. coli ST405 and ST410 carrying carbapenemase (NDM-5) and AmpC (CMY-2), respectively, both located on IncF plasmid highlight the need of strict adherence to infection prevention and control policies by healthcare workers to prevent further dissemination within HCM.Early detection of ESBL, AmpC and carbapenemase genes would be important for the reduction of mortality rate and spread of MDR organisms in studied hospitals. Although the study found evidence of antimicrobial resistance of ExPEC and other GNB, the data collected did not represent the complete picture of the situation in the whole studied hospitals due to small number of samples related to blood and CFS culture contaminations.