Image dataset of urine test results on petri dishes for deep learning classification.

Recent advancements in image analysis and interpretation technologies using computer vision techniques have shown potential for novel applications in clinical microbiology laboratories to support task automation aiming for faster and more reliable diagnostics. Deep learning models can be a valuable tool in the screening process, helping technicians spend less time classifying no-growth results and quickly separating the categories of tests that deserve further analysis. In this context, creating datasets with correctly classified images is fundamental for developing and improving such models. Therefore, a dataset of urine test Petri dishes images was collected following a standardized process, with controlled conditions of positioning and lighting. Image acquisition was conducted by applying a hardware chamber equipped with a led lightning source and a smartphone camera with 12 MP resolution. A software application was developed to support image classification and handling. Experienced microbiologists classified the images according to the positive, negative, and uncertain test results. The resulting dataset contains a total of 1500 images and can support the development of deep learning algorithms to classify urine exams according to their microbial growth.

Virulence Profiling and Molecular Typing of Shiga Toxin-Producing E. coli (STEC) from Human Sources in Brazil.

Since no recent data characterizing Shiga toxin-producing E. coli (STEC) from human infections in Brazil are available, the present study aimed to investigate serotypes, stx genotypes, and accessory virulence genes, and also to perform pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST) of 43 STEC strains recovered from 2007 to 2017. Twenty-one distinct serotypes were found, with serotype O111:H8 being the most common. However, serotypes less frequently reported in human diseases were also found and included a hybrid STEC/ETEC O100:H25 clone. The majority of the strains carried stx1a as the sole stx genotype and were positive for the eae gene. Regarding the occurrence of 28 additional virulence genes associated with plasmids and pathogenicity islands, a diversity of profiles was found especially among the eae-harboring strains, which had combinations of markers composed of up to 12 distinct genes. Although PFGE analysis demonstrated genetic diversity between serotypes such as O157:H7, O111:H8, O26:H11, O118:H16, and O123:H2, high genetic relatedness was found for strains of serotypes O24:H4 and O145:H34. MLST allowed the identification of 17 distinct sequence types (STs) with ST 16 and 21 being the most common ones. Thirty-five percent of the strains studied were not typeable by the currently used MLST approach, suggesting new STs. Although STEC O111:H8 remains the leading serotype in Brazil, a diversity of other serotypes, some carrying virulence genes and belonging to STs incriminated as causing severe disease, were found in this study. Further studies are needed to determine whether they have any epidemiological relevance.

Genome and plasmid context of two rmtG-carrying Enterobacter hormaechei isolated from urinary tract infections in Brazil.

OBJECTIVES: Enterobacter hormaechei is an important causative agent of severe infections in critically ill patients. Aminoglycosides are among the main antibiotics for the treatment of E. hormaechei infections, however the development of antimicrobial resistance is an increasing problem. RmtG is a 16S rRNA methyltransferase, a class of enzymes conferring high-level resistance to clinically relevant aminoglycosides. The aim of this study was to characterise the full genetic context of plasmids harbouring the rmtG gene in two aminoglycoside-resistant E. hormaechei isolated in Brazil. METHODS: ThermtG-harbouring plasmids were transferred to an Escherichia coli J53 recipient strain and were fully sequenced using a MiSeq sequencing system. Complete genome assemblies were accomplished using a combination of Newbler v.3.0, SPAdes 3.10.0 and phrap/cross_match programs. Plasmid sequences were annotated using RAST server and were then manually curated using BLAST databases and ISfinder. Easyfig 2.0 was used to map and compare regions of interest containing rmtG in both plasmids. RESULTS: Both isolates carried thermtG gene on an IncA/C plasmid of ˜152kb and ˜235kb, respectively, associated with a Tn3 transposon. The plasmids contain a transfer region as well as genes involved in plasmid stability and resistance to ß-lactams, sulfonamides and quaternary ammonium compounds. One of the plasmids also carried the mrk operon encoding type 3 fimbriae. CONCLUSION: This first detection ofrmtG in E. hormaechei supports the ability for horizontal transfer. The location in complex genetic platforms carried by Tn3 transposons in IncA/C plasmids may facilitate dissemination to other Gram-negative pathogens, further limiting treatment options.

Draft genome sequences of KPC-2- and CTX-M-15-producing Klebsiella pneumoniae ST437 isolated from a clinical sample and urban rivers in Sao Paulo, Brazil.

OBJECTIVES: KPC-producing Klebsiella pneumoniae is considered one of the most worrisome multidrug-resistant micro-organisms in nosocomial infections. It has also been reported in wastewater and urban rivers in the city of Sao Paulo, Brazil. Here we report the draft genome sequences of three KPC-2- and CTX-M-15-producing K. pneumoniae sequence type 437 (ST437) isolates obtained from two urban rivers and from a clinical sample of a patient in Sao Paulo. METHODS: A genomic library was constructed using a Nextera XT Kit. An Illumina platform was used to perform whole-genome sequencing (WGS). RESULTS: WGS of environmental isolates Kp148/PINH-4900 and Kp196/TIET-4200 and clinical isolate Kp314/11 resulted in estimated genome sizes of 5464058, 5437723 and 5319218bp, respectively. Resistome analysis of the environmental and clinical strains revealed the presence of resistance genes to the following antimicrobials in all strains: aminoglycosides [aac(6')-Ib-cr]; ß-lactams (blaOXA-1, blaSHV-11, blaCTX-M-15 and blaKPC-2); fluoroquinolones [aac(6')-Ib-cr, oqxA and oqxB]; fosfomycin (fosAKP); macrolides [mph(A)]; phenicols (catB4); sulfonamides (sul1); and trimethoprim (dfrA30). The tetracycline resistance gene tetA was identified in Kp148/PINH-4900 and Kp314/11 only; the aminoglycoside resistance gene aph(3')-Ia was found only in environmental isolates, and aadA2 only in Kp314/11; and the phenicol resistance gene catA1 was identified only in Kp148/PINH-4900. CONCLUSIONS: The draft genome sequences of these strains help us to elucidate the dissemination of resistance genes in micro-organisms inside and outside the hospital and are useful for further comparisons between clinical and environmental strains.

Draft genome sequence of a KPC-2-producing Klebsiella pneumoniae ST340 carrying blaCTX-M-15 and blaCTX-M-59 genes: a rich genome of mobile genetic elements and genes encoding antibiotic resistance.

OBJECTIVES: Klebsiella pneumoniae is considered an opportunistic pathogen and an important agent of nosocomial and community infections. It presents the ability to capture and harbour several antimicrobial resistance genes and, in this context, the extensive use of carbapenems to treat serious infections has been responsible for the selection of several resistance genes. This study reports the draft genome sequence of a KPC-2-producing K. pneumoniae strain (Kp10) simultaneously harbouring blaCTX-M-15 and blaCTX-M-59 genes isolated from urine culture of a patient with Parkinson's disease. METHODS: Classical microbiological methods were applied to isolate and identify the strain, and PCR and sequencing were used to identify and characterise the genes and the genetic environment. Whole-genome sequencing (WGS) was performed using a Nextera XT DNA library and a NextSeq platform. RESULTS: WGS analysis revealed the presence of 5915 coding genes, 46 RNA-encoding genes and 255 pseudogenes. Kp10 belonged to sequence type 340 (ST340) of clonal complex 258 (CC258) and carried 20 transferable genes associated with antimicrobial resistance, comprising seven drug classes. Although the simultaneous presence of different blaCTX-M genes in the same strain is rarely reported, the blaKPC-2, blaCTX-M-15 and blaCTX-M-59 genes were not associated with the same genetic mobile structure in Kp10. CONCLUSIONS: These results confirm the capacity of K. pneumoniae to harbour several antimicrobial resistance genes. Thus, this draft genome could help in future epidemiological studies regarding the dissemination of clinically relevant resistance genes.

Draft genome sequence of an aminoglycoside-resistant RmtD2-producing Enterobacter cloacae subsp. cloacae ST395 in Brazil.

OBJECTIVES: Enterobacter cloacae has recently emerged as an important agent of nosocomial infections owing to the dissemination of extended-spectrum ß-lactamases and carbapenemases in this species. In this context, a rise in the therapeutic use of aminoglycosides was noticed, followed by the accelerated development of resistance mechanisms. In this study, we report the draft genome sequence of a multidrug-resistant E. cloacae subsp. cloacae strain (Ec2) isolated from an active surveillance culture of a patient with Chagas disease. METHODS: Whole-genome sequencing (WGS) was performed using a Nextera XT DNA library and NextSeq platform. RESULTS: WGS analysis revealed the presence of 5527 coding genes, 62 RNA-encoding genes and 275 pseudogenes. Strain Ec2 belongs to sequence type 395 (ST395) and carries 22 transferable antibiotic resistance genes, comprising eight antimicrobial classes, including the rmtD2 gene conferring high-level aminoglycoside resistance. CONCLUSIONS: This draft genome can be used in comparative genomic analyses with different E. cloacae strains. In addition, it could help at elucidating epidemiological aspects regarding the dissemination of clinically relevant resistance genes.

Transfer of KPC-2 carbapenemase from Klebsiella pneumoniae to Enterobacter cloacae in a patient receiving meropenem therapy.

The horizontal transfer of a plasmid bearing the blaKPC-2 gene from K. pneumoniae to E. cloacae infecting the respiratory tract of a patient during meropenem therapy was elucidated. This finding is particularly worrisome, since these drugs are of last resort for multidrug-resistant Gram-negative pathogens.

Draft genome sequence of an aminoglycoside-resistant RmtG-producing Pseudomonas aeruginosa ST235 isolated from a cystic fibrosis patient.

Cystic fibrosis (CF) patients are often chronically colonised or infected by non-fermenting Gram-negative bacilli, with Pseudomonas aeruginosa being the most prevalent. In this study, we report the draft genome sequence of a multidrug-resistant P. aeruginosa strain belonging to sequence type ST235, isolated from the respiratory tract of a CF patient with chronic colonisation. Whole-genome sequencing analysis revealed a 6.7Mb genome size and the presence of 12 antibiotic resistance genes, including the rmtG gene conferring high-level aminoglycoside resistance, located on the chromosome.

Prevalence of Extended-Spectrum ß-Lactamases CTX-M-8 and CTX-M-2-Producing Salmonella Serotypes from Clinical and Nonhuman Isolates in Brazil.

We characterized extended-spectrum ß-lactamases (ESBL) enzymes among Salmonella strains isolated in Brazil from 2009 to 2014. Salmonella recovered from both clinical and nonhuman (food, poultry, and environment) sources were subjected to antimicrobial susceptibility testing. ß-lactamases genes were detected by polymerase chain reaction/sequencing; plasmid profiles and transferability were assessed by S1-pulsed field gel electrophoresis (PFGE). Genetic diversity was evaluated by XbaI-PFGE. Out of 630 Salmonella strains screened, 46 displayed ESBL phenotype, distributed across 11 different serotypes. blaCTX-M-8 and blaCTX-M-2 genes were detected at frequencies of 47% and 41%, respectively. blaSHV-5 and blaSHV-2 were also detected but in lower frequencies (4%, 2%). blaTEM-1 gene was detected in 22% of the strains. Most of the ESBL genes were transferable by conjugation, and the respective blaESBL gene was detected in the recipient strain, indicating the location of ESBL determinants on transferable plasmids. XbaI-PFGE revealed genomic diversity of Salmonella Typhimurium bearing blaCTX-M-2, blaCTX-M-8, blaTEM-1, and blaSHV-2 genes. Salmonella Muenchen (harboring blaCTX-M-2) and Salmonella Corvallis (blaCTX-M-8 and blaSHV-5) showed clonal relatedness within respective serotypes. Our findings underscore the occurrence of diverse ESBL genes in several Salmonella serotypes, reinforcing the need for continuous surveillance of resistance genes circulating in human and nonhuman sources.

Outbreak of IMP-producing carbapenem-resistant Enterobacter gergoviae among kidney transplant recipients.

OBJECTIVES: The objective of this study was to investigate a prolonged outbreak of carbapenem-resistant Enterobacter gergoviae (CREG) involving kidney transplant recipients (KTRs) between 2009 and 2014. METHODS: A case-control study was undertaken. Controls (n = 52) were selected from CREG-negative KTRs. Surveillance cultures for CREG were collected weekly. Colonization was defined as isolation of CREG from surveillance samples or from clinical specimens, with no evidence of infection. We also investigated infection control practices at the facility. RESULTS: Of 26 identified cases, 13 had had no known contact with another CREG-positive patient before the first positive culture. Seven patients (27%) developed infection. The site most often colonized was the urinary tract. During the study period two clusters were identified, one in 2009 and another in 2013-14. DNA sequencing revealed blaIMP-1 in all CREG tested. No environmental or hand cultures tested positive for CREG. An audit of infection control practices detected flaws in the handling and cleaning of urinary tract devices. Multivariate analysis identified advanced age, ureteral stent use, retransplantation and male gender as risk factors for CREG acquisition. CONCLUSIONS: An outbreak among KTRs caused by an unusual species of MDR bacteria may have resulted from a common source of contamination related to urinary tract devices.

Microbiological characterization of Delftia acidovorans clinical isolates from patients in an intensive care unit in Brazil.

Delftia acidovorans is an opportunistic agent in several types of infections, both in immunocompromised and immune-competent individuals; its resistance to aminoglycosides and polymyxin, choice drugs for empirical treatment of Gram-negative infections, is remarkable. We report the antimicrobial susceptibility and the genetic relatedness of 24 D. acidovorans strains recovered from tracheal aspirates of 21 adult inpatients hospitalized in an intensive care unit at a Brazilian hospital, from 2012 to 2013. All of the isolates were recovered as pure cultures and in counts above 1,000,000 CFU/mL. None of them were susceptible to polymyxin B, amikacin, gentamicin, or tobramycin; quinolones and trimethoprim-sulfamethoxazole presented varied activities against the isolates, while ß-lactam resistance was not detected. Four clusters were verified in pulsed-field gel electrophoresis analysis, and a major pulsotype comprised 10 strains. A possible, but undetermined common source, can be responsible for this strain dissemination, underscoring the need of reinforcing the adherence to disinfection and infection control standard techniques.

Diversidade genética de Klebsiella pneumoniae produtora de KPC isolada de diversos hospitais do Estado de São Paulo / Genetic diversity of KPC - producing Klebsiella pneumoniae isolated from several hospitals in São Paulo State, Brazil

KPC (Klebsiella pneumoniae carbapenemase) é uma enzima que confere resistência a todos os beta-lactâmicos incluindo os carbapenêmicos. Surtos de K. pneumoniae produtora de KPC foram primeiramente descritos em Nova York (2004) e se disseminaram, relatos indicam que estas bactérias produtoras de KPC são prevalentes no mundo. Em 2005, foi isolada pela primeira vez no Brasil uma cepa de Klebsiella pneumoniae produtora de KPC, e a partir de 2009 se disseminou para vários hospitais no Estado de São Paulo, assim como para outros Estados. O objetivo deste estudo foi analisar a diversidade genética de cepas de K. pneumoniae produtoras de KPC isoladas de vários hospitais do Estado de São Paulo através das técnicas de MLST e PFGE. Um total de 100 cepas de K. pneumoniae, provenientes de vários hospitais do Estado de São Paulo, encaminhadas ao Instituto Adolfo Lutz e previamente confirmadas em nosso laboratório como K. pneumoniae produtora de KPC foram selecionadas e submetidas às técnicas de PFGE e MLST. Foram detectados 13 perfis de PFGE, sendo o perfil A o mais dominante, encontrado em 61% dos isolados. O MLST apresentou 8 sequence type diferentes sendo o ST437 o mais predominante encontrado em 73% dos isolados, seguido pelo ST11 (11%), ST340 (7%), ST258 (3%), ST442 (2%) e ST101 (1%), também foram descritos 2 novos STs, o ST1044 e ST1046. Os dados de PFGE e MLST mostraram como as cepas se disseminaram tanto entre hospitais como entre cidades, aumentando a disseminação dos mecanismos de resistência presentes nesses isolados, como produção de ESBL, carbapenemase e metilases 16S rRNA. Os ST437, ST11, ST340 e ST258, pertencem ao mesmo complexo clonal denominado CC258 disseminado mundialmente e associado com a produção de KPC e CTX-M. Foi observada a predominância de um tipo de clone pertencendo ao ST437 e ao...

KPC (Klebsiella pneumoniae Carbapenemase) is an enzyme that confers resistance to all beta-lactams, including carbapenems. Klebsiella pneumoniae-producing KPC causing nosocomial outbreaks were first reported in New York (2004) and nowadays this KPC-producing microorganism is disseminated worldwide. KPC-producing K. pneumoniae isolated in 2005 was first described in Brazil and from 2009 has spread through several hospitals in Sao Paulo State, as well as other states. The aim of this study was to evaluate the genetic diversity of KPC-producing K. pneumoniae isolates from several hospitals in Sao Paulo State performing MLST and PFGE to detect clonal spread within and among hospitals. A total of 100 isolates of K. pneumoniae from several hospitals in Sao Paulo State sent to Adolfo Lutz Institute and previously confirmed in our laboratory as KPC producers were selected and subjected to Pulsed Field Gel Eletrophoresis (PFGE) and Multi-Locus Sequence Typing (MLST). Eleven PFGE profiles were detected and the profile A was the prevalent, found in 61% of the strains. MLST showed 8 different sequence types (ST) and the ST437 was predominant in 73% of the isolates, followed by ST11 (11%), ST340 (7%), ST258 (3%), ST442 (2%) E ST101 (1%) and two new STs were described, ST1044 and ST1046. PFGE and MLST data showed how strains are disseminated among hospitals and among cities increasing the dissemination of resistance mechanisms such as ESBL-producing, carbapenemases and 16S rRNA methylases. ST437, ST11, ST340 and ST258 belong to the same clonal complex denominated CC258 that are globally disseminated and associated with the production of KPC and CTX-M. The prevalence of the clone belonging to ST437 and to the PFGE A profile was observed in 59% of the isolates. This may suggest a correlation between PFGE and MLST results, althought it was observed different PFGE profiles in the same ST and different ST in the same PFGE profile...