Staphylococcus capitis Bloodstream Isolates: Investigation of Clonal Relationship, Resistance Profile, Virulence and Biofilm Formation.

Staphylococcus capitis has been recognized as a relevant opportunistic pathogen, particularly its persistence in neonatal ICUs around the world. Therefore, the aim of this study was to describe the epidemiological profile of clinical isolates of S. capitis and to characterize the factors involved in the persistence and pathogenesis of these strains isolated from blood cultures collected in a hospital in the interior of the state of São Paulo, Brazil. A total of 141 S. capitis strains were submitted to detection of the mecA gene and SCCmec typing by multiplex PCR. Genes involved in biofilm production and genes encoding enterotoxins and hemolysins were detected by conventional PCR. Biofilm formation was evaluated by the polystyrene plate adherence test and phenotypic resistance was investigated by the disk diffusion method. Finally, pulsed-field gel electrophoresis (PFGE) was used to analyze the clonal relationship between isolates. The mecA gene was detected in 99 (70.2%) isolates, with this percentage reaching 100% in the neonatal ICU. SCCmec type III was the most prevalent type, detected in 31 (31.3%) isolates and co-occurrence of SCCmec was also observed. In vitro biofilm formation was detected in 46 (32.6%) isolates but was not correlated with the presence of the ica operon genes. Furthermore, biofilm production in ICU isolates was favored by hyperosmotic conditions, which are common in ICUs because of the frequent parenteral nutrition. Analysis of the clonal relationship between the isolates investigated in the present study confirms a homogeneous profile of S. capitis and the persistence of clones that are prevalent in the neonatal ICU and disseminated across the hospital. This study highlights the adaptation of isolates to specific hospital environments and their high clonality.

A Study on the Epidemiological-Molecular Role of Staphylococcus aureus Strains in the Development of Ventilator-Associated Pneumonia in a Tertiary Hospital in Brazil.

This study aimed to explore the molecular epidemiology of Staphylococcus aureus isolated from patients on mechanical ventilation and the participation of virulence factors in the development of ventilator-associated pneumonia (VAP). A prospective cohort study was conducted on patients under mechanical ventilation, with periodic visits for the collection of tracheal aspirates and clinical data. The S. aureus isolates were analyzed regarding resistance profile, virulence, expression of protein A and alpha-toxin using Western blot, clonal profile using PFGE, sequence type using MLST, and characterization and quantification of phenol-soluble modulins. Among the 270 patients in the study, 51 S. aureus strains were isolated from 47 patients. The incidence density of S. aureus and MRSA VAP was 2.35/1000 and 1.96/1000 ventilator days, respectively; of these, 45% (n = 5) were resistant to oxacillin, with 100% (n = 5) harboring SCCmec types II and IV. The most frequent among the tested virulence factors were icaA, hla, and hld. The clonal profile showed a predominance of sequence types originating from the community. Risk factors for VAP were the presence of solid tumors and the sea gene. In conclusion, patient-related risk factors, together with microbiological factors, are involved in the development of S. aureus VAP, which is caused by the patient's own strains.

Staphylococcus aureus and CA-MRSA Carriage among Brazilian Indians Living in Peri-Urban Areas and Remote Communities.

The emergence of Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections among indigenous populations has been reported. Usually, indigenous communities live in extreme poverty and are at risk of acquiring infections. In Brazil, healthcare inequality is observed in this population. To date, there are no reports of CA-MRSA infections, and no active search for asymptomatic S. aureus carriage has been conducted among Brazilian Indians. The aim of this study was to investigate the prevalence of colonization with S. aureus and CA-MRSA among Brazilian Indians. We screened 400 Indians (from near urban areas and remote hamlets) for S. aureus and CA-MRSA colonization. The isolates were submitted to clonal profiling by pulsed-field gel electrophoresis (PFGE), and selected isolates were submitted to multilocus sequence typing (MLST). Among 931 specimens (nasal and oral) from different indigenous individuals in remote hamlets, S. aureus was cultured in 190 (47.6%). Furthermore, CA-MRSA was found in three isolates (0.7%), all SCCmec type IV. PFGE analysis identified 21 clusters among the S. aureus isolates, and MLST analysis showed a predominance of sequence type 5 among these isolates. Our study revealed a higher prevalence of S. aureus carriage among Shanenawa ethnicity individuals (41.1%). Therefore, ethnicity appears to be associated with the prevalence of S. aureus in these populations.

Molecular Epidemiology of Staphylococcus aureus and MRSA in Bedridden Patients and Residents of Long-Term Care Facilities.

At present, multidrug-resistant microorganisms are already responsible for community-acquired infections. Methicillin-resistant Staphylococcus aureus (MRSA) poses a serious public health risk worldwide because of the rapid spread and diversification of pandemic clones that are characterized by increasing virulence and antimicrobial resistance. The aim of this study was to identify the prevalence and factors associated with nasal, oral and rectal carriage of S. aureus and MRSA in bedridden patients and residents of long-term care facilities for the elderly (LTCFs) in Botucatu, SP, Brazil. Nasal, oral and rectal swab isolates obtained from 226 LTCF residents or home-bedridden patients between 2017 and 2018 were submitted to susceptibility testing, detection of the mecA gene, SCCmec characterization, and molecular typing by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Logistic regression analysis was used to identify risk factors associated with the presence of S. aureus and MRSA. The prevalence of S. aureus and MRSA was 33.6% (n = 76) and 8% (n = 18), respectively. At the nine LTCFs studied, the prevalence of S. aureus ranged from 16.6% to 85.7% and that of MRSA from 13.3% to 25%. Living in an LTCF, male gender, a history of surgeries, and a high Charlson Comorbidity Index score were risk factors associated with S. aureus carriage, while MRSA carriage was positively associated with male gender. This study showed a high prevalence of S. aureus among elderly residents of small (<15 residents) and medium-sized (15−49 residents) LTCFs and a higher prevalence of MRSA in the oropharynx.

Phenotypic and Molecular Characterization of Nonfermenting Gram-Negative Bacilli Causing Peritonitis in Peritoneal Dialysis Patients.

(1) Background: Peritonitis due to nonfermenting Gram-negative bacilli (NF-GNB) is a dramatic complication of peritoneal dialysis (PD) with bad outcomes. Previous studies of PD-related peritonitis due to Pseudomonas species have shown a low-resolution rate, without a high resistance rate to antipseudomonal antibiotics. This suggests that bacterial virulence factors can act and influence peritonitis evolution. This study aimed to describe the microbiological characteristics of NF-GNB causing PD-related peritonitis and analyze their influence on the outcome. (2) Methods: We analyze the 48 isolates from NF-GNB peritonitis, which were stored in our culture collection regarding bacterial resistance, biofilm, and other virulence factors' production, and clonal profile. Additionally, we collected data on treatment and outcomes from patients' clinical registers. (3) Results: The etiologies were species of Pseudomonas (50%), Acinetobacter (36%), and other NF-GNB (14%). There was a high (75%) proportion of biofilm producer lineages. The in vitro susceptibility rate of Pseudomonas spp. to amikacin, ciprofloxacin, and ceftazidime was significantly greater than that of Acinetobacter spp. and other species; however, there was a similar low-resolution rate (<45%) among the episodes attributable to them. Pseudomonas species have a polyclonal profile, while we found a clone of five multiresistant Acinetobacter baumannii over an 8-year interval (2000-2008), which suggest an origin from the healthcare environment. (4) Conclusions: We are not able to identify any predictor of outcome, but it is possible that biofilm and others virulence factors can act in concert and contribute to the bad outcome.

Coagulase-Negative Staphylococci Clones Are Widely Distributed in the Hospital and Community.

Coagulase-negative staphylococci (CoNS) may be considered contaminants when isolated from clinical specimens but may also be a cause of true infection. This study aimed to compare the clonality and SCCmec type of a collection of CoNS isolated from blood cultures of inpatients, nasal swabs of healthy individuals, and patients with chronic wounds, all from the same community, using SCCmec typing, pulsed-field gel electrophoresis (PFGE), and MLST. Staphylococcus epidermidis, exhibited high clonal diversity, but hospital and community clusters were observed. Nosocomial S. epidermidis clones belonged to sequence types ST2, ST6, and ST23. Some Staphylococcus haemolyticus clones were found to circulate in the hospital and community, while Staphylococcus saprophyticus exhibited very high clonal diversity. Staphylococcus lugdunensis, Staphylococcus warneri, and Staphylococcus capitis revealed several isolates belonging to the same clone in the hospital and community. The detection of different SCCmec types within the same cluster indicated high diversity. S. epidermidis was associated with SCCmec I and III, S. haemolyticus with I and II, S. capitis with type V, Staphylococcus hominis with mec complex type A and ccr1, and S. warneri and S. saprophyticus with SCCmec I. The generation of elements and new combinations of cassette genes were highly associated with CoNS isolates, suggesting that SCCmec may not be a good marker of clonality in these bacteria.

Molecular characterization of methicillin-resistant Staphylococcus aureus among insulin-dependent diabetic individuals in Brazil.

BACKGROUND: People with diabetes mellitus, especially insulin-dependent diabetic patients, are a risk group for staphylococcal infections. Asymptomatic infection with Staphylococcus aureus is common and favors dissemination of the microorganism, rendering these individuals a source of infection. This study aimed to characterize the resistance profile, clonal profile and sequence type, as well as to analyze the prevalence and risk factors for nasal and oropharyngeal carriage of methicillin-susceptible (MSSA) and methicillin-resistant S. aureus (MRSA) isolated from insulin-dependent diabetic individuals in the city of Botucatu, SP, Brazil. METHODS: Staphylococcus aureus was collected from the nasopharynx and oropharynx of 312 community-dwelling insulin-dependent diabetic individuals over a period of 3 years (October 2015 to December 2018). The isolates were characterized by susceptibility profiling, detection of the mecA gene, SCCmec typing, and molecular typing by PFGE and MLST. The risk factors associated with S. aureus and MRSA carriage were determined by logistic regression analysis. RESULTS: The overall prevalence of colonization with S. aureus and MRSA was 30.4% and 4.8%, respectively. Fifteen of the 112 S. aureus isolates carried the mecA gene; SCCmec type IV was identified in 10 isolates, SCCmec type I in three, and SCCmec type II in two. Among the 15 resistant isolates (MRSA), four were susceptible to oxacillin/cefoxitin by the disc diffusion method and one MSSA isolate was resistant to sulfamethoxazole/trimethoprim. The analysis of risk factors revealed a protective effect of age and lung disease, while lower-extremity ulcers were a risk factor for S. aureus. For MRSA, only male gender was significantly associated as a risk factor in multivariate analysis. Clonal profile analysis demonstrated the formation of clusters among MRSA isolates from different patients, with the identification of ST5-IV, ST5-I, and ST8-IV. Isolates carrying ST398 were identified among MSSA and MRSA (ST398-IV). CONCLUSION: Our findings reinforce the importance of epidemiological studies of S. aureus carriage, especially in populations at high risk of infections such as diabetics. The data suggest widespread dissemination of MRSA in the population of insulin-dependent diabetic patients studied, as well as the emergence of important lineages among these individuals.

Staphylococcus spp. Isolated from Bovine Subclinical Mastitis in Different Regions of Brazil: Molecular Typing and Biofilm Gene Expression Analysis by RT-qPCR.

Bovine mastitis is mainly caused by bacteria of the genus Staphylococcus spp., which possess different virulence factors, including the capacity for biofilm formation that provides enhanced protection against the action of immune system components and serves as a barrier against the penetration of antimicrobial agents. This study aimed to characterize 181 Staphylococcus spp. Strains-including Staphylococcusaureus and coagulase-negative staphylococci (CoNS) isolated from bovine subclinical mastitis in six Brazilian states-by molecular methods. RT-qPCR was used to verify the expression of genes of the ica operon-mainly responsible for biofilm formation-as well as bap and bhp. Chromosome similarity among the isolates was investigated by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The icaA gene was detected in 79 (43.6%) isolates, icaB in 24 (13.2%), icaC in 57 (31.4%), and icaD in 127 (70.1%). The bap gene was identified in 66 (36.4%) isolates, while the bhp gene was found in nine (4.9%). RT-qPCR confirmed the expression of the icaA gene in 60 (75.9%) isolates, of icaB in six (25%), of icaC in 26 (45.6%), and of icaD in 80 (63%). Clonal typing of the isolates by PFGE permitted the identification of eight Staphylococcusaureus clusters that simultaneously included ≥3 strains, with a similarity of ≥80%. Regarding the other species studied, three clusters were observed for Staphylococcuschromogenes and four clusters for Staphylococcusepidermidis. Only one cluster each was identified for Staphylococcussaprophyticus and Staphylococcussimulans, while the other species did not form any cluster. With respect to MLST, ST126 and ST1 were the prevalent sequence types in S. aureus, while in S.epidermidis all sequence types were different. These results reveal strains with the same evolutionary origin as other isolates, which might cause infections in humans and animals, suggesting their ability to spread between these species.

Detection of the agr System and Resistance to Antimicrobials in Biofilm-Producing S. epidermidis.

The ability of Staphylococcus epidermidis to produce virulence factors, such as biofilm, added to its increased resistance to antimicrobials can cause infections that are difficult to treat. Many staphylococcal virulence factors are under the control of the accessory gene regulator (agr). The objective of this study was to establish the agr locus and susceptibility of biofilm-producing S. epidermidis specimens to antimicrobial agents, through PCR reactions, reverse transcription polymerase chain reaction (RT-PCR), and the determination of minimum inhibitory concentration (MIC), and to analyze the clonal profile of 300 strains isolated from blood culture specimens from inpatients at a University Hospital in Brazil, over a 20-year period by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) techniques. The ica operon expression was shown in 83.6% strains, bhp gene in 11.5%, and aap gene in 32.8%. Oxacillin resistance was detected in 90.1%, while 4.9% showed tigecycline resistance, and intermediate resistance to quinupristin/dalfopristin was identified in 0.4%. Clonal profile determination showed 11 clusters, with the ST2 type determined as the major cluster. The S. epidermidis biofilm producer demonstrated a predominance of agr I locus, oxacillin resistance, and SCCmec III as well as the potential dissemination of pathogenic clones in hospital settings over long periods.

Short communication: ß-Lactam resistance and vancomycin heteroresistance in Staphylococcus spp. isolated from bovine subclinical mastitis.

The use of antimicrobial agents has led to the emergence of resistant bacterial strains over a relatively short period. Furthermore, Staphylococcus spp. can produce ß-lactamase, which explains the survival of these strains in a focus of infection despite the use of a ß-lactam antibiotic. The aim of this study was to evaluate the resistance of Staphylococcus spp. isolated from bovine subclinical mastitis to oxacillin and vancomycin (by minimum inhibitory concentration) and to detect vancomycin heteroresistance by a screening method. We also evaluated ß-lactamase production and resistance due to hyperproduction of this enzyme and investigated the mecA and mecC genes and performed staphylococcal cassette chromosome mec typing. For this purpose, 181 Staphylococcus spp. isolated from mastitis subclinical bovine were analyzed. Using the phenotypic method, 33 (18.2%) of Staphylococcus spp. were resistant to oxacillin. In contrast, all isolates were susceptible to vancomycin, and heteroresistance was detected by the screening method in 13 isolates. Production of ß-lactamase was observed in 174 (96%) of the Staphylococcus spp. isolates. The mecA gene was detected in 8 isolates, all of them belonging to the species Staphylococcus epidermidis, and staphylococcal cassette chromosome mec typing revealed the presence of type I and type IV isolates.

Antimicrobial Resistance Profile of Planktonic and Biofilm Cells of Staphylococcus aureus and Coagulase-Negative Staphylococci.

The objective of the present study was to determine the antimicrobial resistance profile of planktonic and biofilm cells of Staphylococcus aureus and coagulase-negative staphylococci (CoNS). Two hundred Staphylococcus spp. strains were studied, including 50 S. aureus and 150 CoNS strains (50 S. epidermidis, 20 S. haemolyticus, 20 S. warneri, 20 S. hominis, 20 S. lugdunensis, and 20 S. saprophyticus). Biofilm formation was investigated by adherence to polystyrene plates. Positive strains were submitted to the broth microdilution method to determine the minimum inhibitory concentration (MIC) for planktonic and biofilm cells and the minimal bactericidal concentration for biofilm cells (MBCB). Forty-nine Staphylococcus spp. strains (14 S. aureus, 13 S. epidermidis, 13 S. saprophyticus, 3 S. haemolyticus, 1 S. hominis, 3 S. warneri, and 2 S. lugdunensis) were biofilm producers. These isolates were evaluated regarding their resistance profile. Determination of planktonic cell MIC identified three (21.4%) S. aureus strains that were resistant to oxacillin and six (42.8%) that were resistant to erythromycin. Among the CoNS, 31 (88.6%) strains were resistant to oxacillin, 14 (40%) to erythromycin, 18 (51.4%) to gentamicin, and 8 (22.8%) to sulfamethoxazole/trimethoprim. None of the planktonic isolates were resistant to vancomycin or linezolid. MICs were 2-, 4-, 8-, and up to 16-fold higher for biofilm cells than for planktonic cells. This observation was more common for vancomycin and erythromycin. The MBCB ranged from 8 to >256 µg/mL for oxacillin, 128 to >128 µg/mL for vancomycin, 256 to >256 µg/mL for erythromycin and gentamicin, >64 µg/mL for linezolid, and 32/608 to >32/608 µg/mL for sulfamethoxazole/trimethoprim. The results showed considerably higher MICs for S. aureus and CoNS biofilm cells compared to planktonic cells. Analysis of MBCM confirmed that even high concentrations of vancomycin were unable to eliminate the biofilms of S. aureus and CoNS species. Linezolid was the most effective drug in inhibiting staphylococci in the biofilm, without an increase in the MIC, when compared to planktonic cells. None of the isolates were resistant to this drug.

Detection of Enterotoxigenic Potential and Determination of Clonal Profile in Staphylococcus aureus and Coagulase-Negative Staphylococci Isolated from Bovine Subclinical Mastitis in Different Brazilian States.

Epidemiological studies have identified Staphylococcus aureus as the most common agent involved in food poisoning. However, current research highlights the importance of toxigenic coagulase-negative staphylococci (CoNS) isolated from food. The aim of this study was to characterize Staphylococcus spp. isolated from cows with bovine subclinical mastitis regarding the presence of genes responsible for the production of staphylococcal enterotoxins and of the tst-1 gene encoding toxic shock syndrome toxin 1, and to determine the clonal profile of the isolates carrying any of the genes studied. A total of 181 strains isolated in different Brazilian states, including the South, Southeast, and Northeast regions, were analyzed. The sea gene was the most frequent, which was detected in 18.2% of the isolates, followed by seb in 7.7%, sec in 14.9%, sed in 0.5%, see in 8.2%, seg in 1.6%, seh in 25.4%, sei in 6.6%, and ser in 1.6%. The sej, ses, set, and tst-1 genes were not detected in any of the isolates. The typing of the isolates by pulsed-field gel electrophoresis revealed important S. aureus and S. epidermidis clusters in different areas and the presence of enterotoxin genes in lineages isolated from animals that belong to herds located geographically close to each other.

Genetic Determinants of Methicillin Resistance and Virulence among Staphylococcus aureus Isolates Recovered from Clinical and Surveillance Cultures in a Brazilian Teaching Hospital.

Aims. To quantify the presence of SCCmec types and virulence genes among Staphylococcus aureus colonizing and infecting patients from a teaching hospital. Methods. We analyzed 225 and 84 S. aureus isolates recovered from surveillance and clinical cultures, respectively. Strains were studied for the presence and type of SCCmec, as well as for several virulence genes. Univariate and multivariable analysis were performed in order to identify predictors of invasiveness (defined as isolation from clinical cultures). Results. The presence of SCCmec types III (OR, 2.19, 95% CI, 1.08-4.45) and IV (OR, 5.28 95% CI, 1.35-20.63) and of genes coding for exfoliative toxin B (etb, OR, 6.38, 95% CI, 1.48-27.46) and Panton-Valentine leukocidin (pvl, OR, 2.38, 95% CI, 1.16-4.86) was independently associated with invasiveness. Conclusions. SCCmec types III and IV and virulence genes are associated with greater invasiveness of S. aureus. Patients colonized with methicillin-resistant S. aureus, as well as with strains harboring etb or pvl, may be prone to develop invasive disease. Infection-preventing strategies should be more intensively applied to this group.