The Presence of Virulent and Multidrug-Resistant Clones of Carbapenem-Resistant Klebsiella pneumoniae in Southeastern Brazil.

The emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) represents an urgent threat to global public health due to the limited therapeutic options available to control this pathogen. This study aims to analyze the molecular epidemiology, antimicrobial resistance and virulence profile of CRKP isolated from patients at hospitals in Southeastern Brazil. KPC and other beta-lactamase genes were detected in all strains, which were also multidrug-resistant (MDR). In addition, 11 strains showed resistance to last-resort antimicrobials, such as colistin and tigecycline. MLST analysis revealed eight different sequence types (ST11, ST37, ST147, ST340, ST384, ST394, ST437, and ST628), being two (ST628 and ST394) reported for the first time in Brazil. Strains belonging to the clonal complex 258 (CC258) "high-risk clones" were prevalent in this study. The Galleria mellonella model showed the emergence of virulent CRKP strains in the healthcare environment and, suggests that colistin-resistant strains were associated with higher virulence. This study shows the presence of virulent CRKP-MDR strains in hospitals across Southeastern Brazil, and draws attention to the presence of highly virulent emerging CRKP-MDR ST628 strains, showing that virulent and resistant clones can emerge quickly, requiring constant monitoring.

Dissemination of methicillin-resistant Staphylococcus aureus USA300 ST8/PVL- positive in breast infections in a Brazilian region.

We aimed to analyze the molecular epidemiology of 46 methicillin-resistant Staphylococcus aureus (MRSA) isolated from breast infections. The USA300 lineage carrying SCCmecIVa, arginine catabolic mobile element, t008, ST8, and Panton-Valentine leukocidin genes was predominant (93%). This is the first study that describes the spread of the USA300 methicillin-resistant Staphylococcus aureus clone in breast infections in Brazil.

Epigallocatechin gallate has antibacterial and antibiofilm activity in methicillin resistant and susceptible Staphylococcus aureus of different lineages in non-cytotoxic concentrations.

Staphylococcus aureus is an opportunistic agent that can cause a variety of infections, both hospital and community-acquired. Epigallocatechin gallate (EGCG), a flavonoid present in the leaves of Camellia sinensis, has different biological activities, including antimicrobial potential. Here we evaluate the antibacterial and antibiofilm potential of EGCG in nine clinical strains of S. aureus with different genetic profile and antimicrobial susceptibilities. The minimum inhibitory concentrations (MIC) of EGCG ranged from 7.81 to 62.5 µg/mL, and bactericidal activity was observed at 4 times the MIC. Sub-inhibitory concentrations were able to inhibit biofilm production. Concentrations ≤62.5 µg/mL of EGCG were non-cytotoxic for murine macrophages. EGCG significantly reduced the mortality of infected Galleria mellonella larvae with the S. aureus, having shown relevant antibiofilm properties and efficacy in inhibiting the growth of different clinical isolates of S. aureus, thus being a promising substance for the treatment of infections caused by this agent.

Evaluation of antimicrobial potential and surface morphology in thin films of titanium nitride and calcium phosphate on orthodontic brackets.

INTRODUCTION: The goal of this research was to experimentally evaluate the surface morphology and adhesion capacity of Streptococcus mutans (U159) on brackets with thin films of titanium nitride (TN) and of titanium nitride doped with calcium phosphate (TNCP). METHODS: Twenty-four metallic brackets were equally allocated to 3 groups (n = 8), according to the type of covering (no covering, TNCP, and TN). The coatings were deposited by cathodic cage (TNCP and TN groups) and were evaluated by scanning electron microscopy and energy dispersive x-ray spectrometry. The biofilm formation of S. mutans on the surface of brackets was determined by crystal violet assay and subsequent optical density quantification. RESULTS: There was homogeneity on the surface morphology of the tie wing area in all groups, whereas the TNCP group has presented particles in the slot. After 24 hours, a biofilm of S. mutans was formed in all the observed groups. The optical density obtained in all 3 groups was similar (no covering, 0.347 ± 0.042; TNCP, 0.238 ± 0.055; TN, 0.226 ± 0.057), with no statistically relevant difference (P = 0.06). CONCLUSIONS: The thin film of TNCP has altered the surface of the bracket's slot, whereas the coatings of TN and TNCP have not altered the superficial morphology of the tie wings. The presence of coatings have not influenced the formation of the S. mutans biofilm on the surface of metallic brackets.

EHEC O111:H8 strain and norovirus GII.4 Sydney [P16] causing an outbreak in a daycare center, Brazil, 2019.

BACKGROUND: This study describes the investigation of an outbreak of diarrhea, hemorrhagic colitis (HC), and hemolytic uremic syndrome (HUS) at a daycare center in southeastern Brazil, involving fourteen children, six staff members, six family members, and one nurse. All bacterial and viral pathogens detected were genetically characterized. RESULTS: Two isolates of a strain of enterohemorrhagic Escherichia coli (EHEC) serotype O111:H8 were recovered, one implicated in a case of HUS and the other in a case of uncomplicated diarrhea. These isolates had a clonal relationship of 94% and carried the stx2a and eae virulence genes and the OI-122 pathogenicity island. The EHEC strain was determined to be a single-locus variant of sequence type (ST) 327. EHEC isolates were resistant to ofloxacin, doxycycline, tetracycline, ampicillin, and trimethoprim-sulfamethoxazole and intermediately resistant to levofloxacin and ciprofloxacin. Rotavirus was not detected in any samples, and norovirus was detected in 46.7% (14/30) of the stool samples, three of which were from asymptomatic staff members. The noroviruses were classified as the recombinant GII.4 Sydney [P16] by gene sequencing. CONCLUSION: In this outbreak, it was possible to identify an uncommon stx2a + EHEC O111:H8 strain, and the most recent pandemic norovirus strain GII.4 Sydney [P16]. Our findings reinforce the need for surveillance and diagnosis of multiple enteric pathogens by public health authorities, especially during outbreaks.

Wide genetic heterogeneity and low antimicrobial resistance of enteroaggregative Escherichia coli isolates from several rural communities.

Enteroaggregative Escherichia coli (EAEC), a highly heterogeneous pathotype of E. coli classified as typical and atypical, are an emerging cause of acute and persistent diarrhea. We aimed to investigate whether population living in rural geographic areas, impacts in the heterogeneity, dissemination and antimicrobial susceptibility of EAEC strains. EAEC isolates (n=73) were analysed for the presence of 23 putative virulence factors, plasmid and antimicrobial resistance profiles, biofilm formation, pulsedfield gel electrophoresis (PFGE) and by multilocus sequence typing (MLST). The agg3A, agg4A, agn43, aap, shf, astA, pet, pic/set1A and sat genes, biofilm forming and antimicrobial resistance were statistically associated with typical EAEC. A low frequency of all isolates was resistant or showed a multidrug-resistance profile. No isolate showed the same plasmid profile. In total, 58 different pulsotypes were observed. Sixteen isolates analysed by MLST belonged to 15 different sequence types (ST) and showed a different PFGE pattern and virulence-gene profile. The fact that the communities are semi-isolated did not impact on the peculiar heterogeneity of EAEC, being characterized as epidemiologically independent strains.

Typical and Atypical Enteroaggregative Escherichia coli Are Both Virulent in the Galleria mellonella Model.

Enteroaggregative Escherichia coli (EAEC) is an emerging pathotype responsible for acute and persistent diarrhea. It can be classified as typical and atypical strains, respectively, based on the presence or absence of the AggR regulon, suggesting a higher virulence for typical EAEC. This study aims to evaluate in the Galleria mellonella model if there are differences in the virulence profiles among clinical strains of typical and atypical EAEC, prototype strains EAEC C1096, 042 and its aggR mutant. The clinical EAEC strains (n = 20) were analyzed for the presence of 22 putative virulence factors of EAEC or extraintestinal E. coli by PCR, as well as phenotypic characteristics of virulence (enzymes, siderophore, and biofilm). The survival of the larvae was analyzed after inoculation of 104-107 CFU/larva; the monitoring of bacterial growth in vivo and hemocyte quantification was determined after inoculation of the prototype strains (105 CFU/larva) at different periods after infection. The strains of typical and atypical EAEC presented the same virulence profile for the larva, regardless of the amount or type of genes and phenotypic aspects of virulence analyzed. In addition, the EAEC 042 aggR mutant strain showed a significant reduction in the mortality of the inoculated larvae compared to the wild-type strain. In conclusion, the results obtained herein demonstrate that the virulence of EAEC seems to be related to the AggR regulon, but not exclusively, and atypical EAEC strains may be as virulent as typical ones in vivo in the G. mellonella model.

Differences in resistance profiles and virulence genes among methicillin-resistant and methicillin-susceptible Staphylococcus aureus of different lineages at a public tertiary hospital.

INTRODUCTION: Staphylococcus aureus is a major nosocomial pathogen that is associated with high virulence and the rapid development of drug resistance. METHODS: We analyzed and compared the antimicrobial resistance, virulence profiles, and molecular epidemiology of 67 S. aureus strains, including 36 methicillin-sensitive (MSSA) and 31 methicillin-resistant (MRSA) strains recovered from a public hospital located in south-eastern Brazil. RESULTS: The clones circulating in this hospital presented a great diversity, and the majority of the strains were related to clones responsible for causing worldwide epidemics: these included USA100 (New York/Japan clone), USA300, and USA600. The 31 MRSA (22 SCCmecII and 9 SCCmecIV) and 36 MSSA strains exhibited low resistance against gentamicin and trimethoprim/sulfamethoxazole. No MRSA strain showed resistance to tetracycline. Virulence gene carriage was more diverse and abundant in MSSA than in MRSA. Of the evaluated adhesion-related genes, ebpS was the most prevalent in both MSSA and MRSA strains. The genes bbp and cna showed a strong association with MSSA strains. CONCLUSIONS: Our findings reinforce the idea that MSSA and MRSA strains should be carefully monitored, owing to their high pathogenic potential.

Low Serum Trough Concentrations and High Vancomycin Minimum Inhibitory Concentration in Methicillin-Sensitive Staphylococcus aureus From Hemodialysis Patients in Brazil.

BACKGROUND: The empiric antimicrobial therapy for bacteremia of long-term hemodialysis (HD) outpatients is currently based on the combination of vancomycin and gentamicin because of the high frequency of isolated Staphylococcus species. The vancomycin trough level range from 15 to 20 mcg/mL is expected for therapeutic success against methicillin-resistant Staphylococcus aureus with vancomycin minimum inhibitory concentration (MIC) ≥1.0 mcg/mL. Despite the availability of clinical practice guidelines for vancomycin therapeutic drug monitoring, these target serum concentrations are not reached in many patients. METHODS: In this study, the authors investigated the vancomycin trough levels in 20 HD patients with S. aureus bacteremia and the antimicrobial susceptibility pattern of 45 S. aureus strains isolated from 45 HD patients. The vancomycin serum concentration was determined by chemiluminescent assay. The MIC was determined by broth microdilution method. RESULTS: None of the HD patients included in this study had vancomycin trough concentrations within the therapeutic range. Also, the vancomycin MIC for most methicillin-sensitive S. aureus isolated from bacteremia was ≥1.0 mcg/mL. CONCLUSIONS: The therapeutic range of vancomycin was not achieved, and vancomycin MIC was surprisingly high in methicillin-sensitive S. aureus.

Differences in resistance profiles and virulence genes among methicillin-resistant and methicillin-susceptible Staphylococcus aureus of different lineages at a public tertiary hospital

Abstract INTRODUCTION Staphylococcus aureus is a major nosocomial pathogen that is associated with high virulence and the rapid development of drug resistance. METHODS We analyzed and compared the antimicrobial resistance, virulence profiles, and molecular epidemiology of 67 S. aureus strains, including 36 methicillin-sensitive (MSSA) and 31 methicillin-resistant (MRSA) strains recovered from a public hospital located in south-eastern Brazil. RESULTS The clones circulating in this hospital presented a great diversity, and the majority of the strains were related to clones responsible for causing worldwide epidemics: these included USA100 (New York/Japan clone), USA300, and USA600. The 31 MRSA (22 SCCmecII and 9 SCCmecIV) and 36 MSSA strains exhibited low resistance against gentamicin and trimethoprim/sulfamethoxazole. No MRSA strain showed resistance to tetracycline. Virulence gene carriage was more diverse and abundant in MSSA than in MRSA. Of the evaluated adhesion-related genes, ebpS was the most prevalent in both MSSA and MRSA strains. The genes bbp and cna showed a strong association with MSSA strains. CONCLUSIONS Our findings reinforce the idea that MSSA and MRSA strains should be carefully monitored, owing to their high pathogenic potential.

Typical and atypical enteroaggregative escherichia coli Are both virulent in the galleria mellonella model

Enteroaggregative Escherichia coli (EAEC) is an emerging pathotype responsible for acute and persistent diarrhea. It can be classified as typical and atypical strains, respectively, based on the presence or absence of the AggR regulon, suggesting a higher virulence for typical EAEC. This study aims to evaluate in the Galleria mellonella model if there are differences in the virulence profiles among clinical strains of typical and atypical EAEC, prototype strains EAEC C1096, 042 and its aggR mutant. The clinical EAEC strains (n = 20) were analyzed for the presence of 22 putative virulence factors of EAEC or extraintestinal E. coli by PCR, as well as phenotypic characteristics of virulence (enzymes, siderophore, and biofilm). The survival of the larvae was analyzed after inoculation of 104–107 CFU/larva; the monitoring of bacterial growth in vivo and hemocyte quantification was determined after inoculation of the prototype strains (105 CFU/larva) at different periods after infection. The strains of typical and atypical EAEC presented the same virulence profile for the larva, regardless of the amount or type of genes and phenotypic aspects of virulence analyzed. In addition, the EAEC 042 aggR mutant strain showed a significant reduction in the mortality of the inoculated larvae compared to the wild-type strain. In conclusion, the results obtained herein demonstrate that the virulence of EAEC seems to be related to the AggR regulon, but not exclusively, and atypical EAEC strains may be as virulent as typical ones in vivo in the G. mellonella model.

Typical and atypical enteroaggregative escherichia coli Are both virulent in the galleria mellonella model

Enteroaggregative Escherichia coli (EAEC) is an emerging pathotype responsible for acute and persistent diarrhea. It can be classified as typical and atypical strains, respectively, based on the presence or absence of the AggR regulon, suggesting a higher virulence for typical EAEC. This study aims to evaluate in the Galleria mellonella model if there are differences in the virulence profiles among clinical strains of typical and atypical EAEC, prototype strains EAEC C1096, 042 and its aggR mutant. The clinical EAEC strains (n = 20) were analyzed for the presence of 22 putative virulence factors of EAEC or extraintestinal E. coli by PCR, as well as phenotypic characteristics of virulence (enzymes, siderophore, and biofilm). The survival of the larvae was analyzed after inoculation of 104–107 CFU/larva; the monitoring of bacterial growth in vivo and hemocyte quantification was determined after inoculation of the prototype strains (105 CFU/larva) at different periods after infection. The strains of typical and atypical EAEC presented the same virulence profile for the larva, regardless of the amount or type of genes and phenotypic aspects of virulence analyzed. In addition, the EAEC 042 aggR mutant strain showed a significant reduction in the mortality of the inoculated larvae compared to the wild-type strain. In conclusion, the results obtained herein demonstrate that the virulence of EAEC seems to be related to the AggR regulon, but not exclusively, and atypical EAEC strains may be as virulent as typical ones in vivo in the G. mellonella model.

Clonal dissemination of vancomycin-resistant Enterococcus faecium ST412 in a Brazilian region

ABSTRACT Vancomycin-resistant Enterococcus faecium (VREfm) has emerged as an important global nosocomial pathogen, and this trend is associated with the spread of high-risk clones. Here, we determined the genetic and phenotypic features of 93 VREfm isolates that were obtained from patients in 13 hospitals in Vitória, Espírito Santo, Brazil, during 2012-2013. All the isolates were vancomycin-resistant and harbored the vanA gene. Only 6 (6.5%) of the VREfm isolates showed the ability to form biofilm. The 93 isolates analyzed belong to a single pulsed-field gel electrophoresis lineage and presented six subtypes. MLST genotyping showed that all VREfm belonged to ST412 (the high-risk clone, hospital-adapted). The present study describes the dissemination of ST412 clone in the local hospitals. The clonal spread of these ST412 isolates in the area we analyzed as well as other hospitals in southeastern Brazil supports the importance of identifying and controlling the presence of these microorganisms in health care-related services.

Clonal dissemination of vancomycin-resistant Enterococcus faecium ST412 in a Brazilian region.

Vancomycin-resistant Enterococcus faecium (VREfm) has emerged as an important global nosocomial pathogen, and this trend is associated with the spread of high-risk clones. Here, we determined the genetic and phenotypic features of 93 VREfm isolates that were obtained from patients in 13 hospitals in Vitória, Espírito Santo, Brazil, during 2012-2013. All the isolates were vancomycin-resistant and harbored the vanA gene. Only 6 (6.5%) of the VREfm isolates showed the ability to form biofilm. The 93 isolates analyzed belong to a single pulsed-field gel electrophoresis lineage and presented six subtypes. MLST genotyping showed that all VREfm belonged to ST412 (the high-risk clone, hospital-adapted). The present study describes the dissemination of ST412 clone in the local hospitals. The clonal spread of these ST412 isolates in the area we analyzed as well as other hospitals in southeastern Brazil supports the importance of identifying and controlling the presence of these microorganisms in health care-related services.

Tetracycline and trimethoprim/sulfamethoxazole at clinical laboratory: can they help to characterize Staphylococcus aureus carrying different SCCmec types?

INTRODUCTION: Methicillin-resistant Staphylococcus aureus (MRSA) can be difficult to detect at the clinical practice. METHODS: We analyzed 140 MRSA isolates from inpatients to correlate the antimicrobial susceptibility with the SCCmec types. RESULTS: Type III (n = 63) isolates were more resistant to ciprofloxacin, clindamycin, cloramphenicol, erythromycin, gentamicin, and rifampin than type IV (n = 65) ones (p < 0.05). Moreover, type IV isolates were susceptible to tetracycline (100%) and trimethoprim/sulfamethoxazole (98%), while type III isolates presented resistance to them. CONCLUSIONS: In regions where these SCCmec types are prevalent, the detection of specific resistant phenotypes could help to predict them, mainly when there are no technical conditions to SCCmec typing.

Tetracycline and trimethoprim/sulfamethoxazole at clinical laboratory: Can they help to characterize Staphylococcus aureus carrying different SCCmec types?

INTRODUCTION: Methicillin-resistant Staphylococcus aureus (MRSA) can be difficult to detect at the clinical practice. METHODS: We analyzed 140 MRSA isolates from inpatients to correlate the antimicrobial susceptibility with the SCCmec types. RESULTS: Type III (n = 63) isolates were more resistant to ciprofloxacin, clindamycin, cloramphenicol, erythromycin, gentamicin, and rifampin than type IV (n = 65) ones (p < 0.05). Moreover, type IV isolates were susceptible to tetracycline (100%) and trimethoprim/sulfamethoxazole (98%), while type III isolates presented resistance to them. CONCLUSIONS: In regions where these SCCmec types are prevalent, the detection of specific resistant phenotypes could help to predict them, mainly when there are no technical conditions to SCCmec typing.

Modification of the Congo red agar method to detect biofilm production by Staphylococcus epidermidis.

Staphylococcus epidermidis in immunocompromised patients can cause bacteremia related to the use of catheter due to biofilm production. There are different phenotypic methods to detect biofilm formation. One method is based on culture in brain heart infusion agar (BHIA) containing sucrose and red Congo dye (original Congo red agar). Our group created a new CRA formula and we have confirmed its capacity to detect biofilm production in 210 S. epidermidis strains, including 76 (36.2%) icaAB gene-positive strains. Other parameters were also evaluated. The new CRA formula that gave the best results was BHIA with sucrose (5%), Congo red (0.08%), NaCl (1.5%), glucose (2%), and vancomycin (0.5 mg/mL) (vancomycin-modified CRA-CRAmod). The CRAmod plus vancomycin may be a promising tool and can help to determine the real participation of S. epidermidis in the infectious process.

Methicillin-resistant Staphylococcus aureus in Rio de Janeiro hospitals: dissemination of the USA400/ST1 and USA800/ST5 SCCmec type IV and USA100/ST5 SCCmec type II lineages in a public institution and polyclonal presence in a private one.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) infections have changed since certain non-multiresistant MRSA lineages have emerged in hospitals. In this study, 99 MRSA isolates, 77 from a public and 22 from a private hospital, were characterized. METHODS: Isolates were tested for antimicrobial susceptibility, whereas staphylococcal chromosomal cassette mec (SCCmec) typing and Panton-Valentine leukocidin genes were assessed by polymerase chain reaction. Pulsed-field gel electrophoresis and multilocus sequence typing analyses were carried out to determine the MRSA lineages. RESULTS: High rates of resistance were found to erythromycin (96%), ciprofloxacin (93%), and clindamycin (90%). The SCCmec types found were as follows: type II (14.2%), III (62.6%), and IV (23.2%). Approximately 85% of type III isolates was related to the Brazilian epidemic clone in both hospitals. For type IV isolates, 94.4% were related to both USA400/ sequence type (ST) 1 and USA800/ST5 lineages in the public hospital, whereas the USA400/ST1, USA800/ST5, USA1100/ST30, and EMRSA (Epidemic MRSA)-15/ST22 lineages were detected in the private hospital. Among the SCCmec II isolates, approximately 85% were related to the USA100/ST5 lineage. Three MRSA isolates were positive to Panton-Valentine leukocidin genes. CONCLUSION: The study showed that there was an emergence of USA400/ST1, USA800/ST5 SCCmec IV, and USA100/ST5 SCCmec II MRSA lineages in both hospitals. There was a dissemination of them in the public hospital and a polyclonal presence of the MRSA isolates in the private hospital. The spread of these lineages can be facilitated by the characteristics of the health institution.

Staphylococcus aureus isolates belonging to different multilocus sequence types present specific virulence gene profiles.

To characterize 73 Staphylococcus aureus isolates from infections in an orthopedic hospital in Rio de Janeiro, we investigated the SCCmec types, the clonality by pulsed field gel electrophoresis and multilocus sequence typing, and the presence of virulence genes. Twenty-eight (38.3%) methicillin-resistant (16 SCCmec type IV and 12 type III) isolates were detected. Most (83.5%) of the isolates were included in five lineages: sequence type (ST) 239 (SCCmecIII), 1, 5, 30, and 1462 (SCCmecIV and/or methicillin-susceptible isolates). Virulence genes fnbB, bbp, and pvl were related to STs 239, 30, and 30/1462, respectively. Isolates from STs 1, 5, and 30 presented specific virulence profiles, irrespective of methicillin resistance.

First report of the bla(OXA-58) gene in a clinical isolate of Acinetobacter baumannii in Rio de Janeiro, Brazil.

Carbapenemase production is an important mechanism of carbapenem resistance among nonfermentative Gram-negative isolates. This study aimed to report the detection of bla(OXA-58) gene in multiresistant clinical isolates of Acinetobacter baumannii recovered from inpatients in a public hospital. Polymerase chain reaction tests were performed to detect the bla(OXA-23-like), bla(OXA-24-like), bla(OXA-58-like) and bla(OXA-51-like) genes. The bla(OXA-58) and bla(OXA-23) genes were detected in one and three isolates, respectively. Sequencing of the bla(OXA-58-like) amplicon revealed 100% identity with the A. baumannii bla(OXA-58) gene listed in the GenBank database. This is the first report of an OXA-58-producing A. baumannii isolate in Rio de Janeiro, Brazil.