Occurrence of clinically relevant antimicrobial resistance genes, including mcr-3 and mcr-7.1, in soil and water from a recreation club.

We researched clinically relevant antimicrobial resistance genes (ARGs) and mobile genetic elements (MGEs) in environmental samples from a recreation club in Brazil. A total of 172 amplicons (105 from soil and 67 from water) of 26 ARGs (20 among the soil and water samples; four only in soil samples; two only in water samples) were detected. Nine MGEs were detected, including plasmids and class 1 integron. The absolute abundance of the mcr-3 gene ranged from 1.12 × 102 to 1.81 × 103 copies/mL-1 in water samples. The rapid spread of mcr-like genes in several sources has generated a huge concern to public health. Accordingly, understanding of antimicrobial resistance, carry out surveillance studies may contribute to tackle antimicrobial resistance. As the environmental samples were collected from a popular recreation club in Brazil, this study points out to the risk and exposure to clinically relevant ARGs, especially to mcr-3 and mcr-7.1 genes.

Appearance of mcr-9, blaKPC, cfr and other clinically relevant antimicrobial resistance genes in recreation waters and sands from urban beaches, Brazil.

The co-occurrence of mcr-like and carbapenemase-encoding genes have been reported mainly in humans and animals, whereas, in the environment, studies are gradually increasing due to the One Health approach. In this study, we investigated antimicrobial resistance genes (ARGs) in water and sand samples from marine environments in Brazil. Total DNA from 56 samples (33 sands and 23 waters) was obtained and 27 different ARGs were detected, highlighting the presence of mcr-9, blaKPC and cfr genes. Additionally, the microbiological analysis revealed that sand samples of all analyzed beaches were not recommended for primary use, whereas water samples from most beaches were classified as unsuitable for bathing. The presence of clinically relevant ARGs in urban beaches suggests the presence of antimicrobial-resistant bacteria. Furthermore, to the best of our knowledge, this is the first report of mcr-9 and cfr genes in the environment from Brazil and recreational areas worldwide.

Colistin-Resistant mcr-1-Positive Escherichia coli ST131-H22 Carrying bla CTX-M-15 and qnrB19 in Agricultural Soil.

The pandemic Escherichia coli sequence type 131 (ST131) carrying plasmid-mediated colistin resistance mcr genes has emerged worldwide causing extraintestinal infections, with lineages belonging to three major clades (A, B, and C). Clade B is the most prevalent in animals, contaminating associated meat products, and can be transmitted zoonotically. However, the bla CTX-M-15 gene has only been associated with C2 subclade so far. In this study, we performed a genomic investigation of an E. coli (strain S802) isolated from a kale crop in Brazil, which exhibited a multidrug-resistant (MDR) profile to clinically significant antimicrobials (i.e., polymyxin, broad-spectrum cephalosporins, aminoglycosides, and fluoroquinolones). Whole-genome sequencing analysis revealed that the S802 strain belonged to serotype O25:H4, ST131/CC131, phylogenetic group B2, and virotype D5. Furthermore, S802 carried the clade B-associated fimH22 allele, genes encoding resistance to clinically important antimicrobials, metals, and biocides, and was phylogenetically related to human, avian, and swine ST131-H22 strains. Additionally, IncHI2-IncQ1, IncF [F2:A-:B1], and ColE1-like plasmids were identified harboring mcr-1.1, bla CTX-M-15, and qnrB19, respectively. The emergence of the E. coli ST131-H22 sublineage carrying mcr-1.1, bla CTX-M-15, and qnrB19 in agricultural soil represents a threat to food and environmental safety. Therefore, a One Health approach to genomic surveillance studies is required to effectively detect and limit the spread of antimicrobial-resistant bacteria and their resistance genes.

High Level of Resistance to Antimicrobials and Heavy Metals in Multidrug-Resistant Pseudomonas sp. Isolated from Water Sources.

Antimicrobial-resistant bacteria (ARB) carrying antimicrobial resistance genes (ARGs) have been increasingly detected in water sources. Pseudomonas sp. are opportunistic pathogens commonly reported in water samples and different antimicrobial resistance mechanisms have been described in Pseudomonas sp., producing multidrug-resistant (MDR) phenotype. Besides, the presence of heavy metal resistance genes (HMRGs) may select ARB, which is worrying. Therefore, this study aimed to characterize the genotypes of Pseudomonas sp. isolated from different water sources. Water samples (i.e., rivers, streams, lakes and sewage treatment plants) were collected from different cities in Brazil. The bacterial identification was performed by sequencing the 16S rDNA and the resistance profile to antimicrobials and heavy metals were determined by minimum inhibitory concentration (MIC). Several ARGs, HMRGs, and plasmids were researched by PCR and the amplicons were sequenced for confirmation. A total of 23 Pseudomonas sp. isolates were obtained and identified as Pseudomonas saponiphila, Pseudomonas hunanensis, Pseudomonas aeruginosa, and Pseudomonas asiatica. These isolates presented high MICs to antimicrobials and heavy metals, being the great majority (n = 21; 91%) classified as MDR. Different clinically important ARGs were detected, such as blaGES, qnrS, qepA, tetB, aac(3')-IIa, and ant(2″)-Ia. The czcA gene was the only HMRG detected and no plasmids were found. To the best of our knowledge, this is the first report of the world of P. saponiphila carrying ARGs (i.e., blaGES, qnrS, aac(3')-IIa, tetB) and QepA-producing P. hunanensis and the first time of P. saponiphila, P. asiatica, and P. hunanensis in Brazil.

Co-occurrence of mcr-1, mcr-3, mcr-7 and clinically relevant antimicrobial resistance genes in environmental and fecal samples.

Multidrug-resistant bacteria harboring different antimicrobial resistance genes (ARGs) have been detected worldwide. The association of plasmid-mediated colistin resistance genes (mcr-like) and other ARGs in bacteria isolated from animals is a huge concern worldwide. Therefore, this study aimed to investigate the presence of mcr-like genes and clinically relevant ARGs as well as plasmids in samples from a zoo. Fecal and environmental (soil and water) samples were collected from a zoo and the DNA of cultivable aerobic bacteria was extracted. ARGs were screened by PCR and the plasmids were detected using the PCR-based replicon typing method. A total of 74 amplicons from 27 ARGs [mcr-1, mcr-3, mcr-7.1, blaCTX-M-Gp1, blaCTX-M-Gp2, blaCTX-M-Gp9, blaVEB, blaPER, blaCMY, tetA, tetB, tetC, aadA, aac(6')-Ib, aph(3')-Ia, ant(2'')-Ia, qnrA, qnrB, qnrS, oqxA, oqxB, sul1, sul2, sul3, cmlA, mefAE, ermB] and 21 amplicons from eight plasmid families (IncY, ColE-like, IncFrepB, IncFIA, IncFIB, IncHI1, IncFIC, IncP) were detected. These findings reinforce that the zoo acts as a reservoir of clinically relevant ARGs, including mcr-like, and call attention to the monitoring studies in the zoo. Therefore, to the best of our knowledge, this is the first report of the world of mcr-1, mcr-3 and mcr-7.1 in environmental samples from the zoo.

Occurrence and abundance of clinically relevant antimicrobial resistance genes in environmental samples after the Brumadinho dam disaster, Brazil.

On January 25th 2019, the structure damming a pond containing ore mining wastes and iron burst at Brumadinho City, Brazil. About 11.7 million m3 of a tailings-mud mixture was released from the dam, causing destruction along 300 km of the Paraopeba River toward the São Francisco River. The environments with a high content of metals may provide a suitable environment for horizontal gene transfer, including antimicrobial resistance genes (ARGs). Therefore, this study aimed to detect and quantify clinically relevant ARGs in environmental samples after the Brumadinho dam disaster. Soil, sediment, and water samples were collected within 300 km of the Brumadinho dam disaster at unaffected and affected sites. Physical-chemical parameters of water samples were measured. Total DNA was extracted and 65 clinically relevant ARGs were researched by PCR. The most prevalent ARGs were selected for real-time quantitative PCR analysis. The average of the physical-chemical parameters was higher in the affected sites when compared to the unaffected sites, especially turbidity, concentration of Fe and Al. A total of 387 amplicons from 29 ARGs were detected, which confer resistance to ß-lactams, quinolones, aminoglycosides, tetracyclines, sulphonamides, phenicols, macrolides, glycopeptides, and polymyxins, including extended-spectrum ß-lactamases-encoding genes, and mcr-7.1. The sul1 gene had higher total concentrations than blaTEM, tetB and qnrB in the environmental samples, and the diversity and abundance of ARGs increased at the sites affected by the Brumadinho dam disaster. Therefore, we point out that the contamination by the Brumadinho dam disaster tailings resulted in an increase in the amount and abundance of ARGs in the environment.

Fecal cultivable aerobic microbiota of dairy cows and calves acting as reservoir of clinically relevant antimicrobial resistance genes.

Antimicrobial resistance has become a global threat to public health since multidrug-resistant (MDR) bacteria have been reported worldwide carrying different antimicrobial resistance genes (ARGs), and animals have been described as a reservoir of ARGs. The presence of antimicrobial-resistant bacteria and ARGs in the food matrix is a risk to public health. This study aimed to research the presence of clinically relevant ARGs for important antimicrobials and genetic elements in fecal samples from dairy cows and calves on a Brazilian farm. In this study, a total of 21 fecal samples were collected, and then, the DNA of cultivable aerobic bacteria was extracted. Fifty-seven ARGs and twenty-three genetic elements were researched by PCR and confirmed by sequencing. Several ARGs that confer resistance to ß-lactams, tetracyclines, fluoroquinolones, sulphonamides, phenicols, aminoglycoside, glycopeptides, and macrolides were detected. A total of 200 amplicons from 23 ARGs (blaCTX-M-Gp2, blaCMY, blaSHV, tetA, tetB, tetC, qepA, qnrB, qnrS, oqxA, oqxB, vanC1, vanC2/3, aadA, sul1, sul2, sul3, ermB, mefAE, floR, cmlA, aadA, aph(3')-Ia, aac(3')-Ia), and 145 amplicons from 12 genetic elements (IncF, IncFIA, IncFIB, IncI1, IncY, IncU, IncK, IncP, IncR, IncHI1, ColE-like, intI1) were detected. The results presented in this study call attention to the monitoring of antimicrobial resistance in dairy farms worldwide. MDR bacteria and ARGs can spread to different sources, including milk products, which are one of the most consumed products worldwide, representing a potential risk to human health.

Zinc Supplementation: Immune Balance of Pregnancy During the Chronic Phase of the Chagas Disease.

BACKGROUND: Chagas disease or American trypanosomiasis is caused by the protozoan Trypanosoma cruzi and is endemic of the Americas. The control of the disease is restricted to toxic and potentially teratogenic drugs, which limit the use during pregnancy. The use of food supplementation offers a safe and low-cost form to alleviate Chagas disease symptoms, mostly in areas with alimentary risk. For example, zinc demonstrates positive effects in immune response, including in Chagas disease during pregnancy. PURPOSE: This study describes the innate response in pregnant rats chronically infected with T. cruzi and supplemented with zinc. METHODS: Pregnant female Wistar rats, infected with T. cruzi, were treated with 20 mg/kg/day zinc sulfate and euthanized on the 18th day. Samples (plasma, splenocytes, and peritoneal exudate) were collected and several immune parameters (nitric oxide, RT1B, CD80/CD86, MCP-1, CD11b/c, NK/NKT, IL-2, IL-10, INF-cc, and apoptosis) evaluated. RESULTS: Under Zinc supplementation and/or T. cruzi infection, the gestation developed normally. Several innate immune parameters such as RT1B, CD80/CD86, MCP-1 expressing lymphocytes, IL-2, and IL-17 were positively altered, whereas nitric oxide, CD11b/c, NK/NKT, apoptosis, INF-γ, and corticosterone demonstrated a pro-pregnancy pattern. CONCLUSION: Our results indicated that zinc has diverse effects on immune response during pregnancy. An anti-T. cruzi immunity, as well as a pro-gestation response, were observed after zinc supplementation. The complete comprehension of zinc supplementation in pregnancy will base an adequate strategy to alleviate Chagas disease symptoms and propagation, especially for populations from endemic areas.

Genomic Characterization of a Multidrug-Resistant and Hypermucoviscous/Hypervirulent Klebsiella quasipneumoniae subsp. similipneumoniae ST4417 Isolated from a Sewage Treatment Plant.

Aim: Clinical strains of Klebsiella quasipneumoniae subsp. similipneumoniae have been reported worldwide. Multidrug-resistant (MDR) hypermucoviscous (hm)/hypervirulent (hv) lineages have become a global problem for public health worldwide. Therefore, this study aimed to characterize by whole-genome sequencing a MDR-hm/hv K. quasipneumoniae subsp. similipneumoniae SWT10 strain belonging to the new sequence type ST4417 isolated from a sewage treatment plant. Results: The SWT10 strain was recovered from a sewage treatment plant in Brazil and presented the hm and MDR phenotypes. Resistome analysis showed antimicrobial resistance genes associated with resistance to fluoroquinolones, ß-lactams, tetracyclines, trimethoprim, aminoglycosides, sulfonamides, macrolides, and fosfomycin as well as several heavy metal resistance genes. Virulome analysis showed virulence factors related to hv lineages. Multilocus sequence typing analysis revealed the new ST4417, which was grouped in CC1584 by the minimum-spanning tree. Besides, five plasmid incompatibility groups, two prophage-related sequences, and 66 genomic islands were detected. Conclusion: This study reports for the first time the genome sequence of a MDR-hm/hv K. quasipneumoniae subsp. similipneumoniae recovered from the environment, which contributes to a better understanding about these lineages as well as for surveillance studies worldwide.

Molecular characterization of multidrug-resistant Shiga toxin-producing Escherichia coli harboring antimicrobial resistance genes obtained from a farmhouse.

Shiga toxin-producing Escherichia coli (STEC) colonize the gastrointestinal tract of animals; however, STEC may also cause severe diarrheal diseases. Food-producing animals have been acting as reservoirs and disseminators of multidrug-resistant (MDR) bacteria and antimicrobial resistance genes (ARGs); however, there are few studies characterizing molecularly bacterial isolates from sheep. Therefore, this study aimed to characterize E. coli isolates obtained from feces of sheep in a Brazilian farmhouse. A total of 14 MDR E. coli isolates were obtained from 100 feces samples, six of which were classified as non-O157 STEC (stx1, stx2 and ehxA). MDR E. coli isolates presented different ARGs [blaCTX-M-Gp9, blaCMY, blaSHV, qnrS, oqxB, aac(6')-Ib, tet(A), tet(B), tet(C), sul1, sul2, and cmlA] and plasmids (IncI1, IncFrepB, IncFIB, IncFIA, IncHI1, IncK, and ColE-like). In addition, mutations in the quinolone-resistance determining region of GyrA (Ser83Leu; Asp87Asn) and ParC (Glu84Asp) were detected. PFGE showed a high genetic diversity (30.9 to 83.9%) and thirteen STs were detected (ST25, ST48, ST155, ST162, ST642, ST1247, ST1518, ST1725, ST2107, ST2522, ST3270, ST5036, and ST7100). Subtyping of the fimH gene showed seven fimH-type (25, 32, 38, 41, 54, 61, and 366). The results found in the present study showed high genetic diversity among MDR ARGs-producing E. coli obtained from a farmhouse. This study reports for the first time, the presence of MDR STEC and non-STEC belonging to ST25, ST162, ST642, ST1247, ST1518, ST1725, ST2107, ST3270, ST5036, and ST7100 in sheep, and contributes to the surveillance studies associated with One Health concept.

Characterization of non-O157 Shiga toxin-producing Escherichia coli (STEC) obtained from feces of sheep in Brazil.

Shiga toxin-producing Escherichia coli (STEC) are zoonotic pathogens and may induce severe diarrheagenic diseases in humans and other animals. Non-O157 STEC have been emerging as important pathogens causing outbreaks worldwide. Bacterial resistance to antimicrobials has become a global public health problem, which involves different ecological spheres, including animals. This study aimed to characterize the resistance to antimicrobials, plasmids and virulence, as well as the serotypes and phylogenetic groups in E. coli isolated from sheep in Brazil. A total of 57 isolates were obtained and showed different antimicrobial resistance profiles. Nineteen isolates presented acquired antimicrobial resistance genes (ARGs) (blaCTX-M-Gp9, qnrB, qnrS, oqxB, oqxA, tetA, tetB, tetC, sul1 and sul2) and plasmid families (F, FIA, FIB, I1, K, HI1 and ColE-like). The stx1, stx2 and ehxA virulence genes were detected by PCR, being 50 isolates (87.7%) classified as STEC. A great diversity of serotypes was detected, being O176:HNM the most predominant. Phylogenetic group E was the most prevalent, followed by B1, A and B2. To the best of our knowledge, this is the first report in the world of blaCTX-M-Gp9 (O75, O114, O100, O128ac and O176 serogroups), qnrB and oqxB genes in non-O157 STEC in healthy sheep. The results obtained in the present study call attention to the monitoring of antimicrobial-resistant non-O157 STEC harboring acquired ARGs worldwide and indicate a zoonotic risk due to the profile of virulence, resistance and serotype found.

Heavy metal resistance genes and plasmid-mediated quinolone resistance genes in Arthrobacter sp. isolated from Brazilian soils.

Arthrobacter sp. are Gram-positive bacilli commonly obtained from soil and in the hospital environment. These species have been reported to cause several types of infection. Heavy metals are a threat to the ecological system due to their high-levels of toxicity and the fluoroquinolones are antimicrobials widely used for the treatment of different bacterial infections. The aim of this study was to investigate the resistance to fluoroquinolone and heavy metals, the presence of plasmid-mediated resistance (PMQR) genes and heavy metals resistance (HMR) genes and the presence of plasmids in Arthrobacter sp. obtained from Brazilian soils. Bacterial isolation was performed using soil samples from different Brazilian regions. The bacterial identification was performed by 16S rRNA gene sequencing. The resistance profile for fluoroquinolones and heavy metals was determined by MIC. Several PMQR and HMR genes and plasmid families were investigated by PCR. Eight isolates were obtained from soil samples from different cultivations and regions of Brazil. All isolates were resistant to all fluoroquinolones, cadmium, cobalt and zinc and the majority to copper. Among the PMQR genes, the qepA (4) was the most prevalent, followed by qnrS (3), qnrB (3), oqxB (2) and oqxA (1). Among the HMR genes, the copA was detected in all isolates and the czcA in two isolates. The replication origin of the ColE-like plasmid was detected in all isolates; however, no plasmid was detected by extraction. The association of resistance to heavy metals and antimicrobials is a threat to the environmental balance and to human health. There are no studies reporting the association of PMQR and HMR genes in bacteria belonging to the genus Arthrobacter. To the best of our knowledge, this is the first report of qnrB, qepA, oqxA and oqxB in Arthrobacter species.

Evaluation of different molecular and phenotypic methods for identification of environmental Burkholderia cepacia complex.

The correct identification of different genera and bacterial species is essential, especially when these bacteria cause infections and appropriate therapies need to be chosen. Bacteria belonging to the Burkholderia cepacia complex are considered important opportunistic pathogens, causing different types of infections in immunocompromised, principally in patients with cystic fibrosis. Twenty-one isolates were obtained from different soil samples and identified by sequencing of 16S rRNA, 23S rRNA, recA gene, MLST and by VITEK 2 and MALDI-TOF MS systems. Then, statistical analyses were performed. VITEK 2 and MALDI-TOF MS systems showed different bacterial genera. Sequencing of the 16S rRNA, 23S rRNA gene and amplification of recA gene showed that all the isolates belong to the B. cepacia complex. Sequencing of the recA gene showed a predominance of B. cenocepacia. The PCR of the recA gene showed a high specificity when it is necessary to identify the bacteria belonging to the B. cepacia complex in comparison with 16S and 23S rRNA genes sequencing. MLST analyzes showed a diversity of STs, which have not yet been correlated to the species. Phenotypic identification was not suitable for the identification of these pathogens since in many cases different genera have been reported, including identification by using MALDI-TOF MS.

Characterization of Acquired Antimicrobial Resistance Genes in Environmental Stenotrophomonas maltophilia Isolates from Brazil.

Stenotrophomonas maltophilia is an opportunist pathogen that has intrinsic resistance to the majority of antibiotics and has a high ability to adapt in different environments; however, there are few reports of acquired resistance genes in S. maltophilia. The aim of this study was to investigate the antimicrobial resistance profile, the presence of mutations in the quinolone-resistance determining region, the presence of acquired resistance genes, and the different plasmid families in S. maltophilia isolated from Brazilian soils. A total of 16 isolates were obtained from a variety of agricultural soils with different cultures of Brazil and they were nonsusceptible to most of the antibiotics tested. No mutations were detected in the gyrA gene and only one (Ser-80-Ile) was detected in the parC gene. A diversity of acquired resistance genes was found, including the qnrA, qnrB, qnrS, oqxA, oqxB, blaSHV, blaCTX-M-Gp1, blaPER, blaOXA-1-like, blaOXA-48-like, and sul1. All isolates presented ColE-like plasmids and only one presented IncL/M. These results show, for the first time, the presence of qnrA and oqxAB genes and the presence of qnrB and qnrS genes for the second time in the world in S. maltophilia.

Replicon typing of plasmids in environmental Achromobacter sp. producing quinolone-resistant determinants.

This study aimed to investigate the antimicrobial resistance profile to quinolones, the presence of quinolone-resistant determinants and the plasmid replicon typing in environmental Achromobacter sp. isolated from Brazil. Soil and water samples were used for bacterial isolation. The antimicrobial susceptibility testing was performed by minimum inhibitory concentration method. The detection of mutations in the quinolone resistance-determining regions (QRDR) genes, the presence of plasmid-mediated quinolone resistance (PMQR) genes, and plasmid replicons were performed by PCR. A total of 16 isolates was obtained from different cultures, cities, and states of Brazil. All isolates were non-susceptible to ciprofloxacin, norfloxacin, and levofloxacin. Some mutations in QRDR genes were found, including Gln-83-Leu and Asp-87-Asn in the gyrA and Gln-80-Ile and Asp-84-Ala in the parC. Different PMQR genes were detected, such as qnrA, qnrB, qnrS, oqxA, and oqxB. Three different plasmid families were detected, being most presented the ColE-like, followed by IncFIB and IncA/C. The presence of different PMQR genes and plasmids in the isolates of the present study shows that environmental bacteria can act as reservoir of important genes of resistance to fluoroquinolones, which is of great concern, due to the potential of horizontal dissemination of these genes. Besides that, there are no studies reporting these results in Achromobacter sp. isolates.