Carlos Henrique Camargo

Diversity of Staphylococcus species and prevalence of enterotoxin genes isolated from milk of healthy cows and cows with subclinical mastitis

The objectives of this study were to determine the occurrence and diversity of Staphylococcus spp. in milk from healthy cows and cows with subclinical mastitis in Brazil and to examine the profile of enterotoxin genes and some enterotoxins produced by Staphylococcus spp. A total of 280 individual mammary quarter milk samples from 70 healthy cows and 292 samples from 73 cows with subclinical mastitis were collected from 11 farms in the state of São Paulo, Brazil. Staphylococcus spp. were recovered from 63 (22.5%) samples from healthy cows and from 80 samples (27.4%) from cows with mastitis. The presence of Staphylococcus aureus was significantly different between these 2 groups and was more prevalent in the cows with mastitis. The presence of Staphylococcus saprophyticus was also significantly different between these 2 groups, but this organism was more prevalent in healthy cows. No statistically significant differences were observed in the numbers of other staphylococci in milk samples from the 2 groups. The sea gene was the most prevalent enterotoxin gene in both groups. Eight of 15 (53.3%) Staph. aureus carried this gene and all produced the SEA toxin. In the coagulase-negative staphylococci (CNS) group, 61 of 128 (47.5%) had the same gene and just 1 (1.6%) Staphylococcus epidermidis strain produced the enterotoxin in vitro. Because CNS were isolated from both groups of cows and most CNS contained enterotoxin genes but did not produce toxins, the role of CNS in mastitis should be carefully defined.

Atypical enteropathogenic Escherichia coli as aetiologic agents of sporadic and outbreak-associated diarrhoea in Brazil

Enteropathogenic Escherichia coli (EPEC) are important agents of diarrhoea in industrialized as well as developing countries, such as Brazil. The hallmark of EPEC pathogenesis is the establishment of attaching and effacing lesions in enterocytes, in which pedestal-like structures are formed underneath adherent bacteria. EPEC are divided into two subgroups, typical (tEPEC) and atypical (aEPEC), based on the presence of the EPEC adherence factor plasmid in tEPEC and its absence in aEPEC. This study was designed to characterize 82 aEPEC isolates obtained from stool samples of diarrhoeic patients during 2012 and 2013 in Brazil. The majority of the aEPEC were assigned to the phylo-group B1 (48.8 %), and intimin subtypes θ (20.7 %), β1 (9.7 %) and λ (9.7 %) were the most prevalent among the isolates. The nleB and nleE genes were concomitantly detected in 32.9 % of the isolates, demonstrating the occurrence of the pathogenicity island O122 among them. The O157-plasmid genes (ehxA and/or espP) were detected in 7.3 % of the isolates, suggesting that some aEPEC could be derived from Shiga-toxin-producing E. coli that lost the stx genes while trafficking in the host. PFGE of 14 aEPEC of serotypes O2 : H16, O33 : H34, O39 : H9, O108 : H- and ONT : H19 isolated from five distinct outbreaks showed serotype-specific PFGE clusters, indicating a high degree of similarity among the isolates from the same event, thus highlighting these serotypes as potential aetiologic agents of diarrhoeal outbreaks in Brazil.

Population Structure Analysis of Carbapenem-Resistant Acinetobacter baumannii Clinical Isolates from Brazil Reveals Predominance of Clonal Complexes 1, 15, and 79

ABSTRACT The population structure of 71 carbapenem-resistant Acinetobacter baumannii clinical isolates from several hospitals in Brazil was investigated by ApaI pulsed-field gel electrophoresis, blaOXA-51-like subtyping, and multilocus sequence typing (Institute Pasteur scheme). In addition to the predominance of strains carrying blaOXA-23, we detected the presence of blaOXA-72 and blaOXA-231. We observed a predominance of clonal complex 1 (CC1), CC15, and CC79 and representative strains of the worldwide-disseminated international clone I.

Carbapenem-resistant Enterobacteriaceae Acquired before Liver Transplantation: Impact on Recipient Outcomes.

Background Carbapenem-resistant Enterobacteriaceae (CRE) is an emergent microorganism of infections after liver transplant (LT). The aim of this study was to analyze the risk factors for CRE acquisition and infection after LT. Methods This was a prospective cohort study involving patients who underwent LT in the 2010 to 2014 period. Surveillance cultures for CRE were collected immediately before LT and weekly thereafter until hospital discharge. Results We analyzed 386 patients undergoing a total of 407 LTs. Before LT, 68 (17.6%) patients tested positive for CRE, 11 (16.2%) of those patients having CRE infection, whereas 119 (30.8%) patients acquired CRE after LT. Post-LT CRE infection was identified in 59 (15.7%) patients: Klebsiella pneumoniae was isolated in 83.2%; surgical site infection was the most common type of infection (46.7%). Multivariate analysis showed that post-LT dialysis was the only risk factor for post-LT CRE acquisition. Eighty-two percent of patients who underwent 3 or more post-LT dialysis sessions and acquired CRE before LT evolved with post-LT CRE infection. Other risk factors for CRE infection were acquisition of CRE post-LT, Model for End-Stage Liver Disease score greater than 32, combined transplantation, and reoperation. Patients who acquired CRE before LT had a high risk of developing CRE infection (P < 0.001). Conclusions Measures for minimizing that risk, including altering the antibiotic prophylaxis, should be investigated and implemented.

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 β-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.

Molecular epidemiology of methicillin-susceptible Staphylococcus aureus (MSSA) isolated from milk of cows with subclinical mastitis

Bovine mastitis has been a concern for dairy herd for decades. The adaptation capacity of one of the main species responsible for this disease, Staphylococcus aureus (S. aureus), plays a pivotal role in this issue. The aim of this study was to establish a molecular and phenotypic profile of 285 S. aureus strains isolated from milk of subclinical mastitis cows from 18 different farms in São Paulo State using spa typing, multilocus sequence typing (MLST), pulsed field gel electrophoresis (PFGE), agr cluster (I, II, III and IV) typing, PCR for genes including enterotoxins (sea, seb, sec, sed, see, seg, seh, sei), toxic shock syndrome toxin (tsst-1), and Panton-Valentine leucocidin (pvl), as well as in vitro resistance assays for 12 antibiotics. The results showed a wide variety of strains with a high toxigenic potential; concomitantly, sec, seg and seh were prevalent. In addition, we observed a predominance of the spa types t605 (ST 126, CC126) and t127 (ST1, CC1) and the unusual presence of t321 causing bovine mastitis, which has been previously reported only in swine. The most frequent ST were ST126 (70.5%) and ST1 (10.5%). Regarding PFGE, we observed four major groups and six profile patterns. The highest resistance was observed for streptomycin (9.5%), followed by tetracycline (3.5%), clindamycin (9.3%), and erythromycin (2.8%). The tsst-1 gene was detected in 36.8% of isolates and pvl was not observed. One hundred and thirty-six (47.7%) isolates possessed agr type II, followed by types III (20%) and I (8.1%), with type IV not being detected. We observed that the same spa type could result in different PFGE profiles, so the exclusive use of spa type sequences can lead to incorrect interpretations regarding the spread of clones in an epidemiological context.

Draft Genome Sequence of a Pathogenic Vibrio vulnificus Strain Isolated in Brazil

ABSTRACT We describe the draft genome sequence of the clinical Vibrio vulnificus strain 03_7315, isolated in 2016 from the blood of a diabetic patient who died of septicemia after ingestion of seafood. The draft genome, with 4,755,588 bp covering two chromosomes, presented 4,434 genes, 4,213 coding sequences, and 117 pseudogenes.

In vitro activity of antimicrobial agents against multidrug- and extensively drug-resistant Acinetobacter baumannii.

Acinetobacter baumannii is a leading cause of complicated infections in the hospital environment, particularly among severely ill patients [1]. The crude mortality attributed to Acinetobacter species causing bloodstream infections is higher than 50% [2]. Carbapenems have been one of the main antimicrobial classes used against A. baumannii infections [3], but the emergence and dissemination of carbapenemases have diminished the utility of this class of drugs from an already limited list of existing treatment options [4]. In Brazil, it is known that carbapenem-resistant A. baumannii is considered endemic [5], and the main lineages circulating in that country are those belonging to the clonal complexes (CCs) CC1, CC15 and CC79 [6, 7]. Among the alternatives for the treatment of infections due to carbapenem-resistant A. baumannii, polymyxins and other non-blactam agents, such as tetracyclines, may be valuable options [3]. Given the increasing rates of multidrug(MDR) and extensively drug-resistant (XDR) A. baumannii [8], determination of the antimicrobial susceptibility of alternative agents is needed to assess the availability of potential therapeutic options. Thus, the aim of this study was to assess the in vitro activity of antimicrobial agents against carbapenemresistant A. baumannii isolates recovered from clinical specimens of patients from several hospitals in the state of S~ao Paulo, Brazil.

Evaluation of a new trilocus sequence-based multiplex-PCR to detect major Acinetobacter baumannii clonal complexes circulating in Brazil

Dissemination of carbapenem-resistant Acinetobacter baumannii (CRAB) is mainly due to the spread of clonal lineages, particularly those included into the clonal complexes (CC) CC1, CC2, CC15, CC25, and CC79. We evaluated the usefulness of a recently modified PCR-based trilocus sequence-based typing (m3LST) in comparison with the standard multilocus sequence typing (MSLT) of 7 housekeeping genes as per the Institute Pasteur Scheme to assign the clonal complexes in CRAB. A collection of 78 CRAB isolated from 67 different Brazilian health institutions was submitted to both methodologies, and concordance rate was calculated. The collection studied included mainly isolates belonging to endemic Brazilian Clonal Complexes (CC1, CC15, CC25 and CC79, n=72, 92.3%) but also singletons sequence types (ST) with low prevalence in the country (ST107, ST113, ST188, ST317, ST584, ST733, n=6; 7.7%). The m3LST correctly assigned all the isolates into the main CC responsible for the CRAB dissemination in Brazil. All the singletons ST were not misidentified as prevalent lineages. The PCR-based m3LST is a powerful tool to investigate molecular epidemiology of A. baumannii representative of prevalent Brazilian clonal complexes 1, 15, 25 and 79.

In vitro biofilm production by Staphylococcus spp. strains isolated from finger-foods and snacks

Staphylococcus aureus is one of the most important food-borne pathogens, and although Brazilian Sanitation Surveillance Agency (ANVISA) does not specifically regards for coagulase-negative Staphylococcus (CNS) isolation from foods, it is known that this group of bacteria possesses genes associated with biofilm formation and enterotoxins production. In this context, the present study aimed at identifying the S. aureus and CNS in finger-foods and snacks samples, and to evaluate the ability of these strains to produce biofilm in vitro by means of two methodologies: Congo Red agar and polystyrene microplates cultures. Twenty-two staphylococcal isolates belonging to eight species were obtained from 122 finger-foods, sandwiches and ready-to-eat (RTE) food products. S. aureus, S. warneri and S. haemolyticus were the most frequent isolates. Biofilm production by Staphylococcus spp. was observed in seven (31.8 %) isolates by Congo Red agar technique and three (13.6 %) by polyestirene microplate methodology. There was no positive isolate biofilm producer by both methodologies. Despite the low number of isolates, a concordance of 59.1 % between the tests was found. The ability to produce biofilm is an important virulence factor in Staphylococcus spp., and it can support to define the role of CNS as food-borne pathogen.