Spontaneous fermentation mitigates the frequency of genes encoding antimicrobial resistance spreading from the phyllosphere reservoir to the diet.

The phyllosphere microbiome of vegetable products constitutes an important reservoir for multidrug resistant bacteria and Antibiotic Resistance Genes (ARG). Vegetable products including fermented products such as Paocai therefore may serve as a shuttle for extrinsic microorganisms with ARGs into the gut of consumers. Here we study the effect of fermentation on Paocai ARG dissemination by metagenomic analysis. Microbial abundance and diversity of the Paocai microbiome were diminished during fermentation, which correlated with the reduction of abundance in ARGs. Specifically, as fermentation progressed, Enterobacterales overtook Pseudomonadales as the predominant ARG carriers, and Lactobacillales and Enterobacteriales became the determinants of Paocai resistome variation. Moreover, the dual effect of microbes and metal resistance genes (MRGs) was the major contributor driving Paocai resistome dynamics. We recovered several metagenome-assembled genomes (MAGs) carrying acquired ARGs in the phyllosphere microbiome. ARGs of potential clinical and epidemiological relevance such as tet M and emrB-qacA, were mainly hosted by non-dominant bacterial genera. Overall, our study provides evidence that changes in microbial community composition by fermentation aid in constraining ARG dispersal from raw ingredients to the human microbiome but does not eliminate them.

Nanopore sequencing for identification and characterization of antimicrobial-resistant Escherichia coli and Salmonella spp. from tilapia and shrimp sold at wet markets in Dhaka, Bangladesh.

Wet markets in low-and middle-income countries are often reported to have inadequate sanitation resulting in fecal contamination of sold produce. Consumption of contaminated wet market-sourced foods has been linked to individual illness and disease outbreaks. This pilot study, conducted in two major wet markets in Dhaka city, Bangladesh during a 4-month period in 2021 aimed to assess the occurrence and characteristics of Escherichia coli and non-typhoidal Salmonella spp. (NTS) from tilapia (Oreochromis niloticus) and shrimp (Penaeus monodon). Fifty-four individuals of each species were collected. The identity of the bacterial isolates was confirmed by PCR and their susceptibility toward 15 antimicrobials was tested by disk diffusion. The whole genome of 15 E. coli and nine Salmonella spp. were sequenced using Oxford Nanopore Technology. E. coli was present in 60-74% of tilapia muscle tissue and 41-44% of shrimp muscle tissue. Salmonella spp. was found in skin (29%) and gills (26%) of tilapia, and occasionally in muscle and intestinal samples of shrimp. The E. coli had several Multilocus sequence typing and serotypes and limited antimicrobial resistance (AMR) determinants, such as point mutations on glpT and pmrB. One E. coli (BD17) from tilapia carried resistance genes for beta-lactams, quinolones, and tetracycline. All the E. coli belonged to commensal phylogroups B1 and A and showed no Shiga-toxin and other virulence genes, confirming their commensal non-pathogenic status. Among the Salmonella isolates, five belonged to Kentucky serovar and had similar AMR genes and phenotypic resistance patterns. Three strains of this serovar were ST198, often associated with human disease, carried the same resistance genes, and were genetically related to strains from the region. The two undetermined sequence types of S. Kentucky were distantly related and positioned in a separate phylogenetic clade. Two Brunei serovar isolates, one Augustenborg isolate, and one Hartford isolate showed different resistance profiles. This study revealed high fecal contamination levels in tilapia and shrimp sold at two main wet markets in Dhaka. Together with the occurrence of Salmonella spp., including S. Kentucky ST198, a well-known human pathogen, these results stress the need to improve hygienic practices and sanitation standards at markets to improve food safety and protect consumer health.

Comparative genomic analysis of Aeromonas dhakensis and Aeromonas hydrophila from diseased striped catfish fingerlings cultured in Vietnam.

Introduction: Motile Aeromonas septicemia (MAS) is a burden for striped catfish (Pangasius hypophthalmus) farmers in Vietnam. MAS can be caused by several species of Aeromonas but Aeromonas hydrophila is seen as the leading cause of MAS in aquaculture, but recent reports suggest that A. dhakensis is also causing MAS. Methods: Here we investigated the bacterial etiology of MAS and compared the genomic features of A. hydrophila and A. dhakensis. We collected 86 isolates from diseased striped catfish fingerlings over 5 years from eight provinces in Vietnam. Species identification was done using PCR, MALDI-TOF and whole genome sequence (WGS). The MICs of commonly used antimicrobials was established. Thirty presumed A. hydrophila isolates were sequenced for species confirmation and genomic comparison. A phylogenetic analysis was conducted using publicly available sequences and sequences from this study. Results: A total of 25/30 isolates were A. dhakensis sequence type (ST) 656 and 5/30 isolates were A. hydrophila ST 251. Our isolates and all publicly available A. hydrophila isolates from Vietnam belonged to ST 251 and differed with <200 single nucleotide polymorphisms (SNP). Similarly, all A. dhakensis isolates from Vietnam belonged to ST 656 and differed with <100 SNPs. The tet(A) gene was found in 1/5 A. hydrophila and 19/25 A. dhakensis. All A. hydrophila had an MIC ≤2 mg/L while 19/25 A. dhakensis had MIC ≥8 mg/L for oxytetracycline. The floR gene was only found in A. dhakensis (14/25) which showed a MIC ≥8 mg/L for florfenicol. Key virulence genes, i.e., aerA/act, ahh1 and hlyA were present in all genomes, while ast was only present in A. dhakensis. Discussion: This study confirms previous findings where A. dhakensis was the dominating pathogen causing MAS and that the importance of A. hydrophila has likely been overestimated. The differences in antimicrobial susceptibility between the two species could indicate a need for targeted antimicrobial treatment plans. The lipopolysaccharide regions and outer membrane proteins did not significantly differ in their immunogenic potentials, but it remains to be determined with in vivo experiments whether there is a difference in the efficacy of available vaccines against A. hydrophila and A. dhakensis.

Distribution and ecology of the generalist lactic acid bacterium Carnobacterium maltaromaticum in different freshwater habitats: Metabolic and antagonistic abilities.

We explored the distribution, metabolic and antagonistic activities of Carnobacterium maltaromaticum, isolated from freshwater locations in Denmark during winter or early spring. This species was widely distributed in such habitats although it was relatively rare in low pH locations. Isolates possessed a diverse metabolism, potentially enabling functional capacities independent of habitat. The intraspecies competition showed a relatively high degree of mostly low-intensity interactions, which overall were not correlated with phylogeny or location. Only a few isolates exhibited broad-spectrum inhibition activity, targeting species from other genera and families, including one isolate that exhibited a broad inhibitory activity due to H2 O2 production. Bioinformatic analyses revealed that the frequency of bacteriocinogenic systems was low, and only one unmodified bacteriocin, piscicolin 126, correlated with phenotypic antagonistic activity. Furthermore, most potential bacteriocin gene complexes were not complete. Overall, this study showed C. maltaromaticum to be a generalist (nomadic) species with a constant presence in freshwater habitats, especially those with pH values >5. General metabolic properties did not suggest a strong degree of adaptation to the freshwater environment, and bacteriocin-mediated antagonistic activities appeared to play a minimal ecological role.

Whole strains vs MGEs in short and longterm transmission of ESBL genes between healthcare and community settings in Uganda.

Multidrug-resistant ESBL-producing Escherichia coli are a leading cause of infections in hospital and community settings. Based on samples from two hospitals in Uganda and households of inpatients we tested the hypothesis that ESBL E. coli and/or their resistance determinants could spread within the healthcare and community settings through discharged patients that were still colonized. We used bacterial culture, susceptibility testing whole genome sequencing and detailed bioinformatics analysis to test the above hypothesis. Genome analysis revealed presence of predominantly blaCTX-M-15 and blaOXA-1 genes with a total resistome with genes belonging to 14 different classes of antimicrobials. Short-term cases of strain sharing were reported within each setting and strains from the two settings were found to cluster together based on their overall resistome. Long-term horizontal transfer of ESBL genes by various IncF and IncY types of plasmids shared between healthcare and community settings was demonstrated. Based on hybrid assembly, plasmid reconstruction and phylogenetic analyses, our study suggests that while the dissemination of AMR between healthcare and community settings in the short-term is possible at whole strain level, the long-term transmission between healthcare and communities is sustained by the transfer of plasmids circulating across niches and disseminating related resistomes.

High Genetic Diversity of Carbapenem-Resistant Acinetobacter baumannii Isolates Recovered in Nigerian Hospitals in 2016 to 2020.

Acinetobacter baumannii causes difficult-to-treat infections mostly among immunocompromised patients. Clinically relevant A. baumannii lineages and their carbapenem resistance mechanisms are sparsely described in Nigeria. This study aimed to characterize the diversity and genetic mechanisms of carbapenem resistance among A. baumannii strains isolated from hospitals in southwestern Nigeria. We sequenced the genomes of all A. baumannii isolates submitted to Nigeria's antimicrobial resistance surveillance reference laboratory between 2016 and 2020 on an Illumina platform and performed in silico genomic characterization. Selected strains were sequenced using the Oxford Nanopore technology to characterize the genetic context of carbapenem resistance genes. The 86 A. baumannii isolates were phylogenetically diverse and belonged to 35 distinct Oxford sequence types (oxfSTs), 16 of which were novel, and 28 Institut Pasteur STs (pasSTs). Thirty-eight (44.2%) isolates belonged to none of the known international clones (ICs). Over 50% of the isolates were phenotypically resistant to 10 of 12 tested antimicrobials. The majority (n = 54) of the isolates were carbapenem resistant, particularly the IC7 (pasST25; 100%) and IC9 (pasST85; >91.7%) strains. blaOXA-23 (34.9%) and blaNDM-1 (27.9%) were the most common carbapenem resistance genes detected. All blaOXA-23 genes were carried on Tn2006 or Tn2006-like transposons. Our findings suggest that a 10-kb Tn125 composite transposon is the primary means of blaNDM-1 dissemination. Our findings highlight an increase in blaNDM-1 prevalence and the widespread transposon-facilitated dissemination of carbapenemase genes in diverse A. baumannii lineages in southwestern Nigeria. We make the case for improving surveillance of these pathogens in Nigeria and other understudied settings. IMPORTANCE Acinetobacter baumannii bacteria are increasingly clinically relevant due to their propensity to harbor genes conferring resistance to multiple antimicrobials, as well as their ability to persist and disseminate in hospital environments and cause difficult-to-treat nosocomial infections. Little is known about the molecular epidemiology and antimicrobial resistance profiles of these organisms in Nigeria, largely due to limited capacity for their isolation, identification, and antimicrobial susceptibility testing. Our study characterized the diversity and antimicrobial resistance profiles of clinical A. baumannii in southwestern Nigeria using whole-genome sequencing. We also identified the key genetic elements facilitating the dissemination of carbapenem resistance genes within this species. This study provides key insights into the clinical burden and population dynamics of A. baumannii in hospitals in Nigeria and highlights the importance of routine whole-genome sequencing-based surveillance of this and other previously understudied pathogens in Nigeria and other similar settings.

Salmonella Heidelberg and Salmonella Minnesota in Brazilian broilers: Genomic characterization of third-generation cephalosporin and fluoroquinolone-resistant strains.

Salmonella serovars Heidelberg and Minnesota encoding antimicrobial resistance to third-generation cephalosporins and fluoroquinolones are often detected in poultry/poultry meat. We analysed the genomes of 10 Salmonella Heidelberg (SH) and 4 Salmonella Minnesota (SM) from faecal isolates of Brazilian poultry. These featured virulent and multidrug-resistant characteristics, with AmpC beta-lactamase (blaCMY-2 ) predominance (9/14), for all SM (4/4) and some SH (3/10) located on IncC plasmid replicons. IncC carrying blaCTX-M-2 was only detected among SH (3/10). Mutation in the gyrA/parC genes was present in all SH, whereas SM harboured parC mutation plus qnrB19 on ColRNAI plasmids (3/4). In silico resistance overall corroborated with phenotypic results. Core genome phylogenies showed close clustering and high similarities between the Brazilian and poultry meat/food isolates from Europe, and to human isolates from European countries with documented import of Brazilian poultry meat. Conjugation assays with SM successfully transferred blaCMY-2 , and qnrB19 to an Escherichia coli recipient. The findings reinforce the ongoing antimicrobial resistance acquisition of SH and Minnesota and the risks for disseminating resistant strains and/or mobile elements which may increasingly affect importing countries and the need for controlling AMR in major poultry-exporting countries like Brazil.

Salmonella Heidelberg and Salmonella Minnesota in Brazilian broilers: Genomic characterization of third-generation cephalosporin and fluoroquinolone-resistant strains

Salmonella serovars Heidelberg and Minnesota encoding antimicrobial resistance to third-generation cephalosporins and fluoroquinolones are often detected in poultry/poultry meat. We analysed the genomes of 10 Salmonella Heidelberg (SH) and 4 Salmonella Minnesota (SM) from faecal isolates of Brazilian poultry. These featured virulent and multidrug-resistant characteristics, with AmpC beta-lactamase (blaCMY-2 ) predominance (9/14), for all SM (4/4) and some SH (3/10) located on IncC plasmid replicons. IncC carrying blaCTX-M-2 was only detected among SH (3/10). Mutation in the gyrA/parC genes was present in all SH, whereas SM harboured parC mutation plus qnrB19 on ColRNAI plasmids (3/4). In silico resistance overall corroborated with phenotypic results. Core genome phylogenies showed close clustering and high similarities between the Brazilian and poultry meat/food isolates from Europe, and to human isolates from European countries with documented import of Brazilian poultry meat. Conjugation assays with SM successfully transferred blaCMY-2 , and qnrB19 to an Escherichia coli recipient. The findings reinforce the ongoing antimicrobial resistance acquisition of SH and Minnesota and the risks for disseminating resistant strains and/or mobile elements which may increasingly affect importing countries and the need for controlling AMR in major poultry-exporting countries like Brazil.

Characterization of Escherichia coli and other bacteria isolated from condemned broilers at a Danish abattoir.

Meat inspection is important to ensure food safety and protect public health. Visual inspection of slaughtered carcasses for pathological changes should be supported by bacteriological analysis to determine whether the entire carcass or parts of it should be condemned. The aim of this study was to determine the bacterial species present in different sample types from condemned broiler carcasses. Furthermore, we investigated the genetic characteristics, zoonotic potential, and relatedness of Escherichia coli, the predominant bacterial species isolated from the carcasses. A total of 400 broiler carcasses condemned because of cellulitis (100), scratches (100), hepatitis (100), and healthy control carcasses (100) were selected. Samples of meat, pathological lesion, and bone marrow of each carcass were obtained for microbial analysis. From the analyzed samples, 469 bacterial isolates were recovered with E. coli accounting for 45.8%, followed by Aeromonas spp. (27.9%), in particular A. veronii. The highest rate of bacterial isolation was observed in carcasses condemned with cellulitis, whereas carcasses with hepatitis had the lowest rate of bacterial isolation. Forty-four E. coli isolates originating from different sample types were selected for whole genome sequencing. A clonal relationship was shown between E. coli from different sample types of the same carcass condemned with cellulitis and scratches. A major clade of E. coli was found in carcasses condemned with cellulitis with isolates containing mdf(A), tet(A), and bla TEM-1B genes that confer resistance to macrolides, tetracycline, and ampicillin, respectively. E. coli in this clade all belonged to ST117 and clustered with E. coli isolates previously collected from dead chickens and carcasses condemned due to cellulitis in Denmark, Finland, and the United Kingdom. Bacterial evaluation results of carcasses condemned with cellulitis, scratches (moderate to severe skin lesion), and acute hepatitis confirmed the need for total condemnation of carcasses with these pathological findings. A similar evaluation should be done for carcasses affected with chronic hepatitis, and minor scratches lesions.

Fecal Shedding of Multidrug Resistant Escherichia coli Isolates in Dogs Fed with Raw Meat-Based Diets in Brazil.

The practice of feeding dogs raw meat-based diets (RMBDs) is growing in several countries, and the risks associated with the ingestion of pathogenic and antimicrobial-resistant Escherichia coli in dogs fed these diets are largely unknown. We characterized E. coli strains isolated from dogs fed either an RMBD or a conventional dry feed, according to the phylogroup, virulence genes, and antimicrobial susceptibility profiles of the bacteria. Two hundred and sixteen E. coli strains were isolated. Dogs fed RMBDs shed E. coli strains from the phylogroup E more frequently and were positive for the E. coli heat-stable enterotoxin 1-encoding gene. Isolates from RMBD-fed dogs were also frequently positive for multidrug-resistant E. coli isolates including extended-spectrum beta-lactamase (ESBL) producers. Whole-genome sequencing of seven ESBL-producing E. coli strains revealed that they predominantly harbored blaCTX-M-55, and two strains were also positive for the colistin-resistant gene mcr-1. These results suggest that feeding an RMBD can affect the dog's microbiota, change the frequency of certain phylogroups, and increase the shedding of diarrheagenic E. coli. Also, feeding an RMBD seemed to be linked with the fecal shedding of multidrug-resistant E. coli, including the spread of strains harboring mobilizable colistin resistance and ESBL genes. This finding is of concern for both animal and human health.

Salmonella Salamae and S. Waycross isolated from Nile perch in Lake Victoria show limited human pathogenic potential.

Non-enterica subspecies of Salmonella enterica are rarely associated with human infections. Paradoxically, food safety legislations consider the entire genus Salmonella as pathogenic to humans. Globally, large amounts of seafoods are rejected and wasted due to findings of Salmonella. To inform better food safety decisions, we investigated the pathogenicity of Salmonella Salamae 42:r- and Salmonella Waycross isolated from Nile perch from Lake Victoria. Genome-wide analysis revealed absence of significant virulence determinants including on key Salmonella pathogenicity islands in both serovars. In epithelial cells, S. Salamae showed a weak invasion ability that was lower than the invH mutant of S. Typhimiurium used as negative control. Similarly, S. Salamae could not replicate inside macrophages. Moreover, intracellular replication in S. Waycross strains was significantly lower compared to the wild type S. Typhimurium. Our findings suggest a low pathogenicity of S. Salamae reinforcing the existing literature that non-enterica subspecies are avirulent. We propose that food legislations and actions taken on findings of Salmonella are revisited to avoid wasting valuable sea- and other foods.

Genetic Comparison of ESBL-Producing Escherichia coli from Workers and Pigs at Vietnamese Pig Farms.

We analyzed and compared genomes of Extended Spectrum Beta-Lactamase (ESBL)-producing Escherichia coli from pigs and pig farm workers at 116 farms in Vietnam. Analyses revealed the presence of blaCTX-M-55, blaCTX-M-27, blaCTX-M-15, blaCTX-M-14, blaCTX-M-3, blaCTX-M-65, blaCTX-M-24, blaDHA-1, and blaCMY2 in both hosts. Most strains from pigs contained quinolones (qnr) and colistin resistance genes (mcr-1 and mcr-3). Isolates predominantly harbored more than one plasmid replicon and some harbored plasmid replicons on the same contigs as the ESBL genes. Five strains from farm workers of ST38 (2), ST69 (1), and ST1722 (2) were classified as either uropathogenic E. coli (UPECHM)/extraintestinal pathogenic E. coli (ExPECJJ) or UPECHM, and the remaining were genetically distinct commensals. A high heterogeneity was found among the ESBL-producing E. coli from pigs and workers, with most isolates belonging to unrelated phylogroups, serogroups, and sequence types with >4046 Single-Nucleotide Polymorphisms-(SNPs). In comparing the genomes of pig isolates to those from humans, it appeared that ESBL-producing E. coli in workers did not predominantly originate from pigs but were rather host-specific. Nevertheless, the occurrence of ESBL-producing E. coli carrying plasmid-mediated colistin and quinolone resistance genes in pigs could represent a potential source for horizontal transmission to humans through food rather than direct contact.

ESBL and AmpC ß-Lactamase Encoding Genes in E. coli From Pig and Pig Farm Workers in Vietnam and Their Association With Mobile Genetic Elements.

Animals are considered important sources of ESBL/AmpC-producing bacteria in humans. We analyzed indications of transfer of ESBL/AmpC genes between pigs and pig farmers in Vietnam by analyzing whole genome sequences of 114 ESBL/AmpC-producing E. coli isolated from the two hosts, and performed conjugation experiments and plasmid profiling to confirm that such transfer could have happened. ESBL-encoding genes detected in pigs and pig farmers included bla CTX-M-55, bla CTX-M-27, bla CTX-M-65, bla CTX-M-15, bla CTX-M-14, bla CTX-M-3, bla CTX-M-24, and bla CARB-2, and AmpC ß-lactamases included bla CMY-2, bla DHA-1, and bla CMY-42. The most frequent ESBL gene, bla CTX-M-55, was carried on plasmid with replicons types IncF, IncX, IncH, IncN, IncR, and IncP. The insertion transposases downstream of the bla CTX-M-55 gene were different in plasmids carried by different strains. The second most detected gene, bla CTX-M-27, is found in a stable genetic arrangement with the same flanking transposons seen across strains, and the gene was located on similar conjugal IncF plasmid types, suggesting a horizontal spread of these plasmids. In three strains, we observed a novel bla CTX-M-27 harboring IncF type of plasmid which had not been reported before. Its closest reference in NCBI was the non-ESBL Salmonella Typhimurium plasmid pB71 that might have experienced an insertion of bla CTX-M-27. Our data also point to an emergence of plasmids co-carrying ESBL genes, mcr genes, quinolones and other antimicrobials resistance determinants, and such plasmids require special attention. Plasmids phylogeny confirmed that the bla CTX-M-55 encoding plasmids varied considerably, while those encoding bla CTX-M-27 were closely related. Plasmids harboring both ESBL genes were confirmed to be conjugative and not to differ in transfer efficacy. The isolates carrying the plasmids, even those with plasmids of similar types, showed wide genetic variation with high number of SNPs, suggesting horizontal spread of plasmids into different clonal lines. Their virulence profiles did not confirm to known pathotypes, suggesting that unrelated commensals are a main reservoir for ESBL and AmpC ß-lactamases in both humans and pigs. Overall, despite evidence of transferability of plasmids in the analyzed strains, our findings do not support that ESBL-producing E. coli from pigs or their ESBL/AmpC encoding plasmids are commonly spread to workers in close contact with the animals.

Genomic insights into Vibrio cholerae O1 responsible for cholera epidemics in Tanzania between 1993 and 2017.

BACKGROUND: Tanzania is one of seven countries with the highest disease burden caused by cholera in Africa. We studied the evolution of Vibrio cholerae O1 isolated in Tanzania during the past three decades. METHODOLOGY/PRINCIPAL FINDINGS: Genome-wide analysis was performed to characterize V. cholerae O1 responsible for the Tanzanian 2015-2017 outbreak along with strains causing outbreaks in the country for the past three decades. The genomes were further analyzed in a global context of 590 strains of the seventh cholera pandemic (7PET), as well as environmental isolates from Lake Victoria. All Tanzanian cholera outbreaks were caused by the 7PET lineage. The T5 sub-lineage (ctxB3) dominated outbreaks until 1997, followed by the T10 atypical El Tor (ctxB1) up to 2015, which were replaced by the T13 atypical El Tor of the current third wave (ctxB7) causing most cholera outbreaks until 2017 with T13 being phylogenetically related to strains from East African countries, Yemen and Lake Victoria. The strains were less drug resistant with approximate 10-kb deletions found in the SXT element, which encodes resistance to sulfamethoxazole and trimethoprim. Nucleotide deletions were observed in the CTX prophage of some strains, which warrants further virulence studies. Outbreak strains share 90% of core genes with V. cholerae O1 from Lake Victoria with as low as three SNPs difference and a significantly similar accessory genome, composed of genomic islands namely the CTX prophage, Vibrio Pathogenicity Islands; toxin co-regulated pilus biosynthesis proteins and the SXT-ICE element. CONCLUSION/SIGNIFICANCE: Characterization of V. cholerae O1 from Tanzania reveals genetic diversity of the 7PET lineage composed of T5, T10 and T13 sub-lineages with introductions of new sequence types from neighboring countries. The presence of these sub-lineages in environmental isolates suggests that the African Great Lakes may serve as aquatic reservoirs for survival of V. cholerae O1 favoring continuous human exposure.

Tilapia (Oreochromis niloticus) as a Putative Reservoir Host for Survival and Transmission of Vibrio cholerae O1 Biotype El Tor in the Aquatic Environment.

Studies have reported the occurrence of Vibrio cholerae in fish but little is known about the interaction between fish and toxigenic V. cholerae as opposed to phytoplankton, which are well-established aquatic reservoirs for V. cholerae. The present study determined the role of tilapia (Oreochromis niloticus) as a reservoir host for survival and transmission of V. cholerae in aquatic environments. Three experiments were performed with one repetition each, where O. niloticus (∼2 g) kept in beakers were inoculated with four V. cholerae strains (5 × 107 cfu/mL). Firstly, infected tilapia were kept in stagnant water and fed live brine shrimp (Artemia salina) larvae daily. Secondly, infected tilapia were kept without feeding and water was changed every 24 h. Thirdly, infected tilapia were fed and water was renewed daily. Infected tilapia and non-infected controls were sacrificed on days 1, 2, 3, 7, and 14 post-inoculation and V. cholerae were enumerated in intestinal content and water. Another experiment assessed the transmission of V. cholerae from infected to non-infected tilapia. The study revealed that El Tor biotype V. cholerae O1 and V. cholerae non-O1 colonized tilapia intestines and persisted at stable concentrations during the second week of the experiment whereas the Classical biotype was undetectable after 1 week. In stagnant water with feeding, V. cholerae counts dropped to 105 cfu/ml in water and from 107 to 104 cfu/intestine in fish after 14 days. When water was renewed, counts in water decreased from 107 to 103 cfu/ml and intestinal counts went from 106 to 102 cfu/intestine regardless of feeding. All strains were transmitted from infected to naïve fish after 24 h of cohabitation. Tilapia like other fish may play an essential role in the survival and dissemination of V. cholerae O1 in aquatic environments, e.g., the seventh pandemic strains mostly. In this study, tilapia were exposed to high concentrations of V. cholerae to ensure initial uptake and follow-up studies with lower doses resembling natural concentrations of V. cholerae in the aquatic environment are needed to confirm our findings.

The impact of inactivation of the purine biosynthesis genes, purN and purT, on growth and virulence in uropathogenic E. coli.

De novo synthesis of purines has been suggested to be an important factor for the pathogenesis of uropathogenic E. coli (UPEC). We analyzed the role of the redundant purine biosynthesis genes purN and purT, responsible for the third step in the purine biosynthesis, during UPEC infection. Growth experiments in M9 (minimal media), MOPS (rich media), filtered urine, and human serum with E. coli UTI89 and ΔpurN, ΔpurT, and ΔpurN/T mutants revealed that UPEC relies on de novo purine synthesis for growth in minimal medium. Mutants in individual genes as well as the double mutant grew equally well as the wild type in urine, rich media, and serum. However, during competition for growth in urine, the wild type UTI89 strain significantly outcompeted the purine auxotrophic ΔpurN/T mutant from late exponential growth phase. Inactivation of purN and/or purT significantly affected UPEC invasion of human bladder cells, but not the intracellular survival. Cytotoxicity levels to bladder cells were also diminished when both purN and purT were deleted, while single gene mutants did not differ from the wild type. When infecting human macrophages, no differences were observed between UTI89 and mutants in uptake, survival or cytotoxicity. Finally, the lack of the pur-gene(s), whether analysed as single or double gene knock-out, did not affect recovery rates after in vivo infection in a mouse model of UTI. These findings suggest that de novo synthesis of purines might be required only when UPEC is fully deprived of nucleotides and when grown in competition with other microorganisms in urine.

Molecular Typing and Antimicrobial Susceptibility of Methicillin-Resistant Staphylococcus aureus Isolated from Bovine Milk in Tanzania.

Methicillin-resistant Staphylococcus aureus (MRSA) in raw milk can be transmitted from animals to humans, and in Tanzania raw milk is sold in local markets and consumed as purchased. This study was performed to determine the molecular characteristics and antimicrobial susceptibility pattern of MRSA strains isolated from raw bovine milk sold at local markets in Tanzania. A total of 117 raw milk samples were cultured on Baird-Parker medium to isolate S. aureus and PCR was used for amplification of gltB gene for S. aureus identification and the presence of mecA gene for methicillin-resistant strains. Coagulase-negative (CN) S. aureus were reconfirmed using tube coagulase, DNase, and API Staph tests. MRSA isolates were spa typed whereas antimicrobial susceptibility testing was performed by the disc diffusion method. Forty-six coagulase positives (CP) and two CN S. aureus were identified. Most strains were resistant to penicillin (72%), and 3 isolates: 2 CN S. aureus and 1 coagulase-negative Staphylococci (CNS), were phenotypically resistant to vancomycin, oxacillin, and cefoxitin and were confirmed to carry mecA. Resistance to clindamycin, trimethoprim-sulfamethoxazole, and tetracycline was 23.9%, 30.4%, and 41.3%, respectively. Twelve isolates exhibited multidrug resistance; however, only one mecA positive strain among the three was typeable and belonged to spa type t2603. This study reports for the first time the presence of CN variant of MRSA, which was assigned the spa type t2603, and the presence of multidrug resistant S. aureus isolates from bovine milk in Morogoro, Tanzania.

Antimicrobial resistance among clinically relevant bacterial isolates in Accra: a retrospective study.

OBJECTIVE: The aim of this study was to determine the antimicrobial resistance pattern of bacterial isolates from different specimens at various hospitals and private diagnostic service laboratories in Ghana. RESULTS: A retrospective data of culture and sensitivity test results from 2016 were extracted from the microbiology record book of six laboratories in Accra, Ghana. The data included type of clinical specimen, sex of patient, name of bacterial isolate and antibiotic resistance profile. A total of 16.6% (n = 10,237) resistant isolates were obtained, however, the proportions of resistant isolates varied significantly between laboratories. High resistance towards tetracycline, ampicillin, cotrimoxazole and cephalosporins, but low towards amoxiclav and aminoglycosides, was observed. This study identified E. coli and Staphylococcus species as the major resistant bacteria from clinical specimen in Accra and the highest prevalence of the isolates was found in urine specimens in all six laboratories (69.1%, n = 204; 52.6%, n = 36; 52.3%, n = 350; 37.9%, n = 298; 53%, n = 219; 62.1%, n = 594) and in female patients (81.4, 50 and 69.5%). Regular surveillance and local susceptibility pattern analysis is extremely important in selecting the most appropriate and effective antibiotic for the treatment of bacterial infections.