Thymidine-dependent Staphylococcus aureus and lung function in patients with cystic fibrosis: a 10-year retrospective case-control study.

OBJECTIVE: Thymidine-dependent small-colony variants (TD-SCVs) of Staphylococcus aureus are being isolated with increasing frequency from patients with cystic fibrosis (CF). The aim of this study was to evaluate the relationship between TD-SCV isolation and pulmonary function in patients with CF, as well as to determine whether the emergence of TD-SCVs was associated with trimethoprim-sulfamethoxazole (TMP-SMX) use and with coinfection with other microorganisms. METHODS: This was a retrospective case-control study including patients with CF who visited the Clinical Hospital Complex of the Federal University of Paraná, in Curitiba, Brazil, between 2013 and 2022. Demographic, clinical, and spirometric data, as well as information on TD-SCVs and other isolated microorganisms, were collected from the medical records of patients with CF and TD-SCVs (TD-SCV group; n = 32) and compared with those of a matched group of patients with CF without TD-SCVs (control group; n = 64). RESULTS: Isolation of TD-SCVs was positively associated with TMP-SMX use (p = 0.009), hospitalization (p < 0.001), and impaired pulmonary function (p = 0.04). CONCLUSIONS: The use of TMP-SMX seems to contribute to the emergence of TD-SCVs, the isolation of which was directly associated with worse pulmonary function in our sample.

Comparative genomics of IncQ1 plasmids carrying blaGES variants from clinical and environmental sources in Brazil.

IncQ-type plasmids have become important vectors in the dissemination of blaGES among different bacterial genera and species from different environments around the world, and studies estimating the occurrence of Guiana extended-spectrum (GES)-type ß-lactamases are gaining prominence. We analyzed the genetic aspects of two IncQ1 plasmids harboring different blaGES variants from human and environmental sources. The blaGES variants were identified using polymerase chain reaction (PCR) in Aeromonas veronii isolated from hospital effluent and Klebsiella variicola isolated from a rectal swab of a patient admitted to the cardiovascular intensive care unit in a different hospital. Antimicrobial-susceptibility testing and transformation experiments were performed for phenotypic analysis. Whole-genome sequencing was performed using Illumina and Oxford Nanopore platforms. The comparative analysis of plasmids was performed using BLASTn, and the IncQ1 plasmids showed a high identity and similar size. A. veronii harbored blaGES-7 in a class 1 integron (In2061), recently described by our group, and K. variicola carried blaGES-5 in the known class 1 integron. Both integrons showed a fused gene cassette that encodes resistance to aminoglycosides and fluoroquinolones, with an IS6100 truncating the 3'-conserved segment. The fused genes are transcribed together, although the attC site is disrupted. These gene cassettes can no longer be mobilized. This study revealed a mobilome that may contribute to the dissemination of GES-type ß-lactamases in Brazil. Class 1 integrons are hot spots for bacterial evolution, and their insertion into small IncQ-like plasmids displayed successful recombination, allowing the spread of blaGES variants in various environments. Therefore, they can become prevalent across clinically relevant pathogens.

Thymidine-dependent Staphylococcus aureus and lung function in patients with cystic fibrosis: a 10-year retrospective case-control study

ABSTRACT Objective: Thymidine-dependent small-colony variants (TD-SCVs) of Staphylococcus aureus are being isolated with increasing frequency from patients with cystic fibrosis (CF). The aim of this study was to evaluate the relationship between TD-SCV isolation and pulmonary function in patients with CF, as well as to determine whether the emergence of TD-SCVs was associated with trimethoprim-sulfamethoxazole (TMP-SMX) use and with coinfection with other microorganisms. Methods: This was a retrospective case-control study including patients with CF who visited the Clinical Hospital Complex of the Federal University of Paraná, in Curitiba, Brazil, between 2013 and 2022. Demographic, clinical, and spirometric data, as well as information on TD-SCVs and other isolated microorganisms, were collected from the medical records of patients with CF and TD-SCVs (TD-SCV group; n = 32) and compared with those of a matched group of patients with CF without TD-SCVs (control group; n = 64). Results: Isolation of TD-SCVs was positively associated with TMP-SMX use (p = 0.009), hospitalization (p < 0.001), and impaired pulmonary function (p = 0.04). Conclusions: The use of TMP-SMX seems to contribute to the emergence of TD-SCVs, the isolation of which was directly associated with worse pulmonary function in our sample.

Low performance of Policimbac® broth microdilution in determining polymyxin B MIC for Klebsiella pneumoniae.

Klebsiella pneumoniae is a global threat to healthcare, and despite the availability of new drugs, polymyxins are still an important therapeutic option for this and other resistant gram-negative pathogens. Broth microdilution is the only method that is recommended for polymyxins. In this study, we evaluated the accuracy of a commercial Policimbac® plate in determining the polymyxin B MIC for K. pneumoniae clinical isolates. The results were compared with those of the broth microdilution method according to ISO 16782. The Policimbac® plate had an excellent 98.04% categorical agreement, but unacceptable 31.37% essential agreement rates. Almost 2% of major errors as observed. Additionally, 52.94% of the strains overestimated the MIC at 1 µg/mL. Three isolates were excluded from the analysis due to the drying of the Policimbac® plate. To avoid dryness, we included wet gauze for the test, obtaining a 100% of categorical agreement rate; however, a low essential agreement was maintained (25.49%). In conclusion, the Policimbac® plate was unable to correctly determine the polymyxin B MIC for K. pneumoniae isolates. This low performance may interfere with the clinical use of the drug and, thus, with the result of the patient's treatment.

In vitro selection of Neisseria gonorrhoeae unveils novel mutations associated with extended-spectrum cephalosporin resistance.

The emergence of Neisseria gonorrhoeae strains resistant to extended-spectrum cephalosporins (ESCs) is a worldwide concern because this class of antibiotics represents the last empirical treatment option for gonorrhea. The abusive use of antimicrobials may be an essential factor for the emergence of ESC resistance in N. gonorrhoeae. Cephalosporin resistance mechanisms have not been fully clarified. In this study, we mapped mutations in the genome of N. gonorrhoeae isolates after resistance induction with cefixime and explored related metabolic pathways. Six clinical isolates with different antimicrobial susceptibility profiles and genotypes and two gonococcal reference strains (WHO F and WHO Y) were induced with increasing concentrations of cefixime. Antimicrobial susceptibility testing was performed against six antimicrobial agents before and after induction. Clinical isolates were whole-genome sequenced before and after induction, whereas reference strains were sequenced after induction only. Cefixime resistance induction was completed after 138 subcultures. Several metabolic pathways were affected by resistance induction. Five isolates showed SNPs in PBP2. The isolates M111 and M128 (ST1407 with mosaic penA-34.001) acquired one and four novel missense mutations in PBP2, respectively. These isolates exhibited the highest minimum inhibitory concentration (MIC) for cefixime among all clinical isolates. Mutations in genes contributing to ESC resistance and in other genes were also observed. Interestingly, M107 and M110 (ST338) showed no mutations in key determinants of ESC resistance despite having a 127-fold increase in the MIC of cefixime. These findings point to the existence of different mechanisms of acquisition of ESC resistance induced by cefixime exposure. Furthermore, the results reinforce the importance of the gonococcal antimicrobial resistance surveillance program in Brazil, given the changes in treatment protocols made in 2017 and the nationwide prevalence of sequence types that can develop resistance to ESC.

Lower airway microbiota and decreasing lung function in young Brazilian cystic fibrosis patients with pulmonary Staphylococcus and Pseudomonas infection.

Cystic fibrosis (CF) is a genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator gene that leads to respiratory complications and mortality. Studies have shown shifts in the respiratory microbiota during disease progression in individuals with CF. In addition, CF patients experience short cycles of acute intermittent aggravations of symptoms called pulmonary exacerbations, which may be characterized by a decrease in lung function and weight loss. The resident microbiota become imbalanced, promoting biofilm formation, and reducing the effectiveness of therapy. The aim of this study was to monitor patients aged 8-23 years with CF to evaluate their lower respiratory microbiota using 16S rRNA sequencing. The most predominant pathogens observed in microbiota, Staphylococcus (Staph) and Pseudomonas (Pseud) were correlated with clinical variables, and the in vitro capacity of biofilm formation for these pathogens was tested. A group of 34 patients was followed up for 84 days, and 306 sputum samples were collected and sequenced. Clustering of microbiota by predominant pathogen showed that children with more Staph had reduced forced expiratory volume in one second (FEV1) and forced vital capacity (FVC) compared to children with Pseud. Furthermore, the patients' clinical condition was consistent with the results of pulmonary function. More patients with pulmonary exacerbation were observed in the Staph group than in the Pseud group, as confirmed by lower body mass index and pulmonary function. Additionally, prediction of bacterial functional profiles identified genes encoding key enzymes involved in virulence pathways in the Pseud group. Importantly, this study is the first Brazilian study to assess the lower respiratory microbiota in a significant group of young CF patients. In this sense, the data collected for this study on the microbiota of children in Brazil with CF provide a valuable contribution to the knowledge in the field.

Different Swine Production Systems Can Shape Slurry Resistome at Mechanism and Class Levels Based on Swine Manure Evaluation.

Antimicrobial resistance is a major threat to public health. Antimicrobial use in animal husbandry is a major concern since it can favor an increase in antimicrobial resistance among farms. Herein, we aim to better understand and characterize the main resistome profiles in microbial communities found in pig farms. Sampling of swine manure was performed in two different timepoints (October 2019 and January 2020) in each of the 14 different swine farms, located in the mesoregion of Western Santa Catarina state in Brazil, a pole of swine product production of worldwide importance. Samples were divided into three groups: farms with the opened regimen and no usage of antimicrobials (F1; n = 10), farms with the closed regimen and usage of antimicrobials (F2; n = 16), and farms with the closed regimen and no usage of antimicrobials (F3; n = 2). The metagenomic evaluation was performed to obtain and identify genetic elements related to antimicrobial resistance using nanopore sequencing. We used ResistoXplorer software to perform composition, alpha and beta diversity, and clustering analysis. In addition, PCR reactions were performed to confirm the presence or absence of seven different beta-lactamase family genes and five phosphoethanolamine transferase gene variants clinically relevant. Our findings based on the identification of resistance genes at the mechanism level showed a prevalence of alteration of the drug target (72.3%) profile, followed by drug inactivation (17.5%) and drug efflux (10.1%). We identified predominantly aminoglycosides (45.3%), tetracyclines (15.9%), and multiclass (11,2%) resistance genes. PCoA analysis indicates differences between F1 and F2 profiles. F2 samples showed increased diversity when compared to the F1 group. In addition, herein we first report the identification of mcr-4 in a slurry sample (C1F1.1) in Santa Catarina State. In general, our findings reinforce that many factors on the practices of animal husbandry are involved in the resistome profile at the mechanism and class levels. Further studies to better understand microbiome and mobilome aspects of these elements are necessary to elucidate transmission pathways between different bacteria and environments.

Novel Insights into blaGES Mobilome Reveal Extensive Genetic Variation in Hospital Effluents.

Mobile genetic elements contribute to the emergence and spread of multidrug-resistant bacteria by enabling the horizontal transfer of acquired antibiotic resistance among different bacterial species and genera. This study characterizes the genetic backbone of blaGES in Aeromonas spp. and Klebsiella spp. isolated from untreated hospital effluents. Plasmids ranging in size from 9 to 244 kb, sequenced using Illumina and Nanopore platforms, revealed representatives of plasmid incompatibility groups IncP6, IncQ1, IncL/M1, IncFII, and IncFII-FIA. Different GES enzymes (GES-1, GES-7, and GES-16) were located in novel class 1 integrons in Aeromonas spp. and GES-5 in previously reported class 1 integrons in Klebsiella spp. Furthermore, in Klebsiella quasipneumoniae, blaGES-5 was found in tandem as a coding sequence that disrupted the 3' conserved segment (CS). In Klebsiella grimontii, blaGES-5 was observed in two different plasmids, and one of them carried multiple IncF replicons. Three Aeromonas caviae isolates presented blaGES-1, one Aeromonas veronii isolate presented blaGES-7, and another A. veronii isolate presented blaGES-16. Multilocus sequence typing (MLST) analysis revealed novel sequence types for Aeromonas and Klebsiella species. The current findings highlight the large genetic diversity of these species, emphasizing their great adaptability to the environment. The results also indicate a public health risk because these antimicrobial-resistant genes have the potential to reach wastewater treatment plants and larger water bodies. Considering that they are major interfaces between humans and the environment, they could spread throughout the community to clinical settings. IMPORTANCE In the "One Health" approach, which encompasses human, animal, and environmental health, emerging issues of antimicrobial resistance are associated with hospital effluents that contain clinically relevant antibiotic-resistant bacteria along with a wide range of antibiotic concentrations, and lack regulatory status for mandatory prior and effective treatment. blaGES genes have been reported in aquatic environments despite the low detection of these genes among clinical isolates within the studied hospitals. Carbapenemase enzymes, which are relatively unusual globally, such as GES type inserted into new integrons on plasmids, are worrisome. Notably, K. grimontii, a newly identified species, carried two plasmids with blaGES-5, and K. quasipneumoniae carried two copies of blaGES-5 at the same plasmid. These kinds of plasmids are primarily responsible for multidrug resistance among bacteria in both clinical and natural environments, and they harbor resistant genes against antibiotics of key importance in clinical therapy, possibly leading to a public health problem of large proportion.

Emergence of Thymidine-Dependent Staphylococcus aureus Small-Colony Variants in Cystic Fibrosis Patients in Southern Brazil.

We characterized Staphylococcus aureus small-colony variant (SCV) strains isolated from cystic fibrosis (CF) patients in southern Brazil. Smaller colonies of S. aureus were isolated from respiratory samples collected consecutively from 225 CF patients from July 2013 to November 2016. Two phenotypic methods-the auxotrophic classification and a modified method of antimicrobial susceptibility testing-were employed. PCR was conducted to detect the mecA, ermA, ermB, ermC, msrA, and msrB resistance genes. Furthermore, DNA sequencing was performed to determine the mutations in the thyA gene, and multilocus sequence typing was used to identify the genetic relatedness. S. aureus strains were isolated from 186 patients (82%); suggestive colonies of SCVs were obtained in 16 patients (8.6%). The clones CC1 (ST1, ST188, and ST2383), CC5 (ST5 and ST221), and ST398 were identified. Among SCVs, antimicrobial susceptibility testing showed that 77.7% of the isolates were resistant to multiple drugs, and all of them were susceptible to vancomycin. mecA (2), ermA (1), ermB (1), ermC (3), and msrB (18) were distributed among the isolates. Phenotypically thymidine-dependent isolates had different mutations in the thyA gene, and frameshift mutations were frequently observed. Of note, revertants showed nonconservative or conservative missense mutations. SCVs are rarely identified in routine laboratory tests. IMPORTANCE Similar findings have not yet been reported in Brazil, emphasizing the importance of monitoring small-colony variants (SCVs). Altogether, our results highlight the need to improve detection methods and review antimicrobial therapy protocols in cystic fibrosis (CF) patients.

Genomic analysis of Neisseria elongata isolate from a patient with infective endocarditis.

Neisseria elongata is part of the commensal microbiota of the oropharynx. Although it is not considered pathogenic to humans, N. elongata has been implicated in several cases of infective endocarditis (IE). Here, we report a case of IE caused by N. elongata subsp. nitroreducens (Nel_M001) and compare its genome with 17 N. elongata genomes available in GenBank. We also evaluated resistance and virulence profiles with Comprehensive Antibiotic Resistance and Virulence Finder databases. The results showed a wide diversity among N. elongata isolates. Based on the pangenome cumulative curve, we demonstrate that N. elongata has an open pangenome. We found several different resistance genes, mainly associated with antibiotic efflux pumps. A wide range of virulence genes was observed, predominantly pilus formation genes. Nel_M001 was the only isolate to present two copies of some pilus genes and not present nspA gene. Together, our results provide insights into how this commensal microorganism can cause IE and may assist further biological investigations on nonpathogenic Neisseria spp. Case reporting and pangenome analyses are critical for enhancing our understanding of IE pathogenesis, as well as for alerting physicians and microbiologists to enable rapid identification and treatment to avoid unfavorable outcomes.

Molecular Epidemiology of Multidrug-Resistant Klebsiella pneumoniae Isolates in a Brazilian Tertiary Hospital.

Multidrug-resistant (MDR) Klebsiella pneumoniae (Kp) is a major bacterial pathogen responsible for hospital outbreaks worldwide, mainly via the spread of high-risk clones and epidemic resistance plasmids. In this study, we evaluated the molecular epidemiology and ß-lactam resistance mechanisms of MDR-Kp strains isolated in a Brazilian academic care hospital. We used whole-genome sequencing to study drug resistance mechanisms and their relationships with a K. pneumoniae carbapenemase-producing (KPC) Kp outbreak. Forty-three Kp strains were collected between 2003 and 2012. Antimicrobial susceptibility testing was performed for 15 antimicrobial agents, and polymerase chain reaction (PCR) was used to detect 32 resistance genes. Mutations in ompk35, ompk36, and ompk37 were evaluated by PCR and DNA sequencing. Pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were carried out to differentiate the strains. Based on distinct epidemiological periods, six Kp strains were subjected to whole-genome sequencing. ß-lactamase coding genes were widely distributed among isolates. Almost all isolates had mutations in porin genes, particularly ompk35. The presence of bla KPC promoted a very high increase in carbapenem minimum inhibitory concentration only when ompk35 and ompk36 were interrupted by insertion sequences. A major cluster was identified by PFGE analysis and all isolates from this cluster belonged to clonal group (CG) 258. We have also identified a large repertoire of resistance genes in the sequenced isolates. A bla KPC-2-bearing plasmid (pUFPRA2) was also identified, which was very similar to a plasmid previously described in the first Brazilian KPC-Kp (2005). We found high-risk clones (CG258) and an epidemic resistance plasmid throughout the duration of the study (2003 to 2012), emphasizing a persistent presence of MDR-Kp strains in the hospital setting. Finally, we found that horizontal transfer of resistance genes between clones may have played a key role in the evolution of the outbreak.

Ralstonia mannitolilytica bacteremia in a neonatal intensive care unit.

Ralstonia mannitolilytica, a Gram-negative bacterium, is rarely isolated in clinical laboratories. It has been associated with outbreaks due to its ability to survive in liquid media and hospital devices. We describe three cases of bacteremia caused by R. mannitolilytica in a neonatal intensive care unit in Curitiba, Southern Brazil. All isolates presented the same PFGE profile. The common source of infection was undetected in surveillance cultures for the outbreak survey. All patients received antimicrobial treatment and were discharged from the maternity. Due to the characteristics of the microorganism, clinicians and microbiologists should pay attention to the emergence of Ralstonia spp. infections.

Ralstonia mannitolilytica bacteremia in a neonatal intensive care unit

Abstract Ralstonia mannitolilytica, a Gram-negative bacterium, is rarely isolated in clinical laboratories. It has been associated with outbreaks due to its ability to survive in liquid media and hospital devices. We describe three cases of bacteremia caused by R. mannitolilytica in a neonatal intensive care unit in Curitiba, Southern Brazil. All isolates presented the same PFGE profile. The common source of infection was undetected in surveillance cultures for the outbreak survey. All patients received antimicrobial treatment and were discharged from the maternity. Due to the characteristics of the microorganism, clinicians and microbiologists should pay attention to the emergence of Ralstonia spp. infections.

Bacteremia and meningitis caused by OXA-23-producing Acinetobacter baumannii - molecular characterization and susceptibility testing for alternative antibiotics.

BACKGROUND: Carbapenem-resistant Acinetobacter baumannii infection is a concern in developing countries due to high incidence, few therapeutic options, and increasing costs. OBJECTIVE: Characterize and analyze the antibiotic susceptibility patterns of carbapenem-resistant A. baumannii isolates and evaluate clinical data of meningitis and bacteremia caused by this microorganism. METHODS: Twenty-six A. baumannii isolates from 23 patients were identified by MALDI-TOF and automated methods and genotyped using pulsed field genotyping electrophoresis. Clinical data and outcomes were evaluated. Susceptibility of isolates to colistin, tigecycline, meropenem, imipenem, and doxycycline was determined. RESULTS: Mortality due to A. baumannii infections was 73.91%; all patients with meningitis and 7/8 patients with ventilator-associated pneumonia died. All isolates were susceptibility to polymyxin (100%; MIC50, MIC90: 1µg/mL, 1µg/mL) and colistin (100%; MIC50, MIC90: 2µg/mL, 2µg/mL), and 92% were susceptible to tigecycline (MIC50, MIC90: 1µg/mL, 1µg/mL) and doxycycline (MIC50, MIC90: 2µg/mL, 2µg/mL). blaOXA-23 was identified in 24 isolates. Molecular typing showed 8 different patterns: 13 isolates belonged to pattern A (50%). CONCLUSION: Carbapenem-resistant A. baumannii infections mortality is high. Alternative antimicrobial therapy (doxycycline) for selected patients with carbapenem-resistant A. baumannii infection should be considered.

Matrix-assisted laser desorption ionization-time of flight: a promising alternative method of identifying the major coagulase-negative Staphylococci species.

INTRODUCTION.: This study aimed to evaluate different methods for differentiation of species of coagulase-negative staphylococci (CoNS) that caused infections in hospitalized immunocompromised patients. METHODS.: A total of 134 CoNS strains were characterized using four different methods. RESULTS.: The results of matrix assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS) analysis were in complete agreement with those of tuf gene sequencing (kappa index = 1.00). The kappa index of Vitek 2® Compact analysis was 0.85 (very good) and that of the conventional method was 0.63 (moderate). CONCLUSIONS: . MALDI-TOF MS provided rapid and accurate results for the identification of CoNS (134; 100%).

Matrix-assisted laser desorption ionization-time of flight: a promising alternative method of identifying the major coagulase-negative Staphylococci species

Abstract INTRODUCTION. This study aimed to evaluate different methods for differentiation of species of coagulase-negative staphylococci (CoNS) that caused infections in hospitalized immunocompromised patients. METHODS. A total of 134 CoNS strains were characterized using four different methods. RESULTS. The results of matrix assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS) analysis were in complete agreement with those of tuf gene sequencing (kappa index = 1.00). The kappa index of Vitek 2® Compact analysis was 0.85 (very good) and that of the conventional method was 0.63 (moderate). CONCLUSIONS . MALDI-TOF MS provided rapid and accurate results for the identification of CoNS (134; 100%).

Bacteremia and meningitis caused by OXA-23-producing Acinetobacter baumannii molecular characterization and susceptibility testing for alternative antibiotics

Abstract Background: Carbapenem-resistant Acinetobacter baumannii infection is a concern in developing countries due to high incidence, few therapeutic options, and increasing costs. Objective: Characterize and analyze the antibiotic susceptibility patterns of carbapenem-resistant A. baumannii isolates and evaluate clinical data of meningitis and bacteremia caused by this microorganism. Methods: Twenty-six A. baumannii isolates from 23 patients were identified by MALDI-TOF and automated methods and genotyped using pulsed field genotyping electrophoresis. Clinical data and outcomes were evaluated. Susceptibility of isolates to colistin, tigecycline, meropenem, imipenem, and doxycycline was determined. Results: Mortality due to A. baumannii infections was 73.91%; all patients with meningitis and 7/8 patients with ventilator-associated pneumonia died. All isolates were susceptibility to polymyxin (100%; MIC50, MIC90: 1 µg/mL, 1 µg/mL) and colistin (100%; MIC50, MIC90: 2 µg/mL, 2 µg/mL), and 92% were susceptible to tigecycline (MIC50, MIC90: 1 µg/mL, 1 µg/mL) and doxycycline (MIC50, MIC90: 2 µg/mL, 2 µg/mL). bla OXA-23 was identified in 24 isolates. Molecular typing showed 8 different patterns: 13 isolates belonged to pattern A (50%). Conclusion: Carbapenem-resistant A. baumannii infections mortality is high. Alternative antimicrobial therapy (doxycycline) for selected patients with carbapenem-resistant A. baumannii infection should be considered.

Bacteremia and meningitis caused by OXA-23-producing Acinetobacter baumannii - molecular characterization and susceptibility testing for alternative antibiotics

Abstract Background: Carbapenem-resistant Acinetobacter baumannii infection is a concern in developing countries due to high incidence, few therapeutic options, and increasing costs. Objective: Characterize and analyze the antibiotic susceptibility patterns of carbapenem-resistant A. baumannii isolates and evaluate clinical data of meningitis and bacteremia caused by this microorganism. Methods: Twenty-six A. baumannii isolates from 23 patients were identified by MALDI-TOF and automated methods and genotyped using pulsed field genotyping electrophoresis. Clinical data and outcomes were evaluated. Susceptibility of isolates to colistin, tigecycline, meropenem, imipenem, and doxycycline was determined. Results: Mortality due to A. baumannii infections was 73.91%; all patients with meningitis and 7/8 patients with ventilator-associated pneumonia died. All isolates were susceptibility to polymyxin (100%; MIC50, MIC90: 1 µg/mL, 1 µg/mL) and colistin (100%; MIC50, MIC90: 2 µg/mL, 2 µg/mL), and 92% were susceptible to tigecycline (MIC50, MIC90: 1 µg/mL, 1 µg/mL) and doxycycline (MIC50, MIC90: 2 µg/mL, 2 µg/mL). bla OXA-23 was identified in 24 isolates. Molecular typing showed 8 different patterns: 13 isolates belonged to pattern A (50%). Conclusion: Carbapenem-resistant A. baumannii infections mortality is high. Alternative antimicrobial therapy (doxycycline) for selected patients with carbapenem-resistant A. baumannii infection should be considered.

Characterization of CTX-M enzymes, quinolone resistance determinants, and antimicrobial residues from hospital sewage, wastewater treatment plant, and river water.

Multidrug-resistant (MDR) bacteria are widespread in hospitals and have been increasingly isolated from aquatic environments. The aim of the present study was to characterize extended-spectrum ß-lactamase (ESBL) and quinolone-resistant Enterobacteriaceae from a hospital effluent, sanitary effluent, inflow sewage, aeration tank, and outflow sewage within a wastewater treatment plant (WWTP), as well as river water upstream and downstream (URW and DRW, respectively), of the point where the WWTP treated effluent was discharged. ß-lactamase (bla) genes, plasmid-mediated quinolone resistance (PMQR), and quinolone resistance-determining regions (QRDRs) were assessed by amplification and sequencing in 55 ESBL-positive and/or quinolone-resistant isolates. Ciprofloxacin residue was evaluated by high performance liquid chromatography. ESBL-producing isolates were identified in both raw (n=29) and treated (n=26) water; they included Escherichia coli (32), Klebsiella pneumoniae (22) and Klebsiella oxytoca (1). Resistance to both cephalosporins and quinolone was observed in 34.4% of E. coli and 27.3% of K. pneumoniae. Resistance to carbapenems was found in 5.4% of K. pneumoniae and in K. oxytoca. Results indicate the presence of blaCTX-M (51/55, 92.7%) and blaSHV (8/55, 14.5%) ESBLs, and blaGES (2/55, 3.6%) carbapenemase-encoding resistance determinants. Genes conferring quinolone resistance were detected at all sites, except in the inflow sewage and aeration tanks. Quinolone resistance was primarily attributed to amino acid substitutions in the QRDR of GyrA (47%) or to the presence of PMQR (aac-(6')-Ib-cr, oqxAB, qnrS, and/or qnrB; 52.9%) determinants. Ciprofloxacin residue was absent only from URW. Our results have shown strains carrying ESBL genes, PMQR determinants, and mutations in the gyrA QRDR genes mainly in hospital effluent, URW, and DRW samples. Antimicrobial use, and the inefficient removal of MDR bacteria and antibiotic residue during sewage treatment, may contribute to the emergence and spreading of resistance in the environment, making this a natural reservoir.

Polyphasic characterisation of Burkholderia cepaciacomplex species isolated from children with cystic fibrosis.

Cystic fibrosis (CF) patients with Burkholderia cepacia complex (Bcc) pulmonary infections have high morbidity and mortality. The aim of this study was to compare different methods for identification of Bcc species isolated from paediatric CF patients. Oropharyngeal swabs from children with CF were used to obtain isolates of Bcc samples to evaluate six different tests for strain identification. Conventional (CPT) and automatised (APT) phenotypic tests, polymerase chain reaction (PCR)-recA, restriction fragment length polymorphism-recA, recA sequencing, and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) were applied. Bacterial isolates were also tested for antimicrobial susceptibility. PCR-recA analysis showed that 36 out of the 54 isolates were Bcc. Kappa index data indicated almost perfect agreement between CPT and APT, CPT and PCR-recA, and APT and PCR-recA to identify Bcc, and MALDI-TOF and recA sequencing to identify Bcc species. The recA sequencing data and the MALDI-TOF data agreed in 97.2% of the isolates. Based on recA sequencing, the most common species identified were Burkholderia cenocepacia IIIA (33.4%),Burkholderia vietnamiensis (30.6%), B. cenocepaciaIIIB (27.8%), Burkholderia multivorans (5.5%), and B. cepacia (2.7%). MALDI-TOF proved to be a useful tool for identification of Bcc species obtained from CF patients, although it was not able to identify B. cenocepacia subtypes.