Multilocus sequence typing of hospital-associated Enterococcus faecium from Brazil reveals their unique evolutionary history

We studied the genetic relationships between vancomycin-susceptible (n = 11) and -resistant Enterococcus faecium (VRE, n = 20) recovered from Brazil using a multilocus sequence typing (MLST) scheme. Grouping of allelic profiles revealed six clusters of related sequence types (STs) that differ in no more than two of the seven alleles. Of these, one cluster harbored 16 of the 20 isolates recovered during the first VRE outbreak in Brazil. The ampicillin and gentamicin resistance profiles were stable in the isolates that clustered within the groups I-III. Comparison with the allelic profiles of 139 E. faecium from different geographical regions and origins found in the international database http://www.mlst.net revealed that the Brazilian outbreak clone did not cluster in the previously named complex-17. This genetic complex contains hospital epidemic and clinical isolates recovered from different countries and continents. Twenty two of the 31 Brazilian isolates, including the VRE outbreak clone, clustered apart from the E. faecium isolates from the database, suggesting that these Brazilian isolates have a distinct evolutionary history.     

Clonal distribution and differential occurrence of the enterotoxin gene cluster, egc, in carriage- versus bacteremia-associated isolates of Staphylococcus aureus

The Staphylococcus aureus enterotoxin gene cluster, egc, was detected in isolates from healthy individuals and in those from patients with bacteremia. The egc genes cooccur and are slightly enriched in strains from healthy carriers (present in 63.7% of carriage-associated isolates versus 52.9% of invasive isolates; P = 0.03). Multilocus sequence typing revealed that successful staphylococcal clones usually harbor the egc locus.     

Sequence typing confirms that Campylobacter jejuni strains associated with Guillain-Barré and Miller-Fisher syndromes are of diverse genetic lineage, serotype, and flagella type

Guillain-Barré syndrome (GBS) and Miller-Fisher syndrome (MFS) are correlated with prior infection by Campylobacter jejuni in up to 40% of cases. Nucleotide sequence-based typing of 25 C. jejuni isolates associated with neuropathy permitted robust comparisons with equivalent data from approximately 800 C. jejuni isolates not associated with neuropathy. A total of 13 genetic lineages and 20 flaA short variable region nucleotide sequences were present among the 25 isolates. A minority of isolates (4 of 25) had the flaA short variable region nucleotide sequences that were previously proposed as a marker for GBS-associated isolates. These 4 isolates probably represented the Penner serotype 19 lineage, which has been proposed to have an association with GBS.     

Accuracy of the VITEK 2 system to detect glycopeptide resistance in enterococci

We evaluated the accuracy of the VITEK 2 fully automated system to detect and identify glycopeptide-resistant enterococci (GRE) compared to a reference agar dilution method. The sensitivity of vancomycin susceptibility testing with VITEK 2 for the detection of vanA, vanB, and vanC1 strains was 100%. The sensitivity of vancomycin susceptibility testing of vanC2 strains was 77%. The sensitivity of teicoplanin susceptibility testing of vanA strains was 90%. Of 80 vanC enterococci, 78 (98%) were correctly identified by VITEK 2 as Enterococcus gallinarum/Enterococcus casseliflavus. Since the identification and susceptibility data are produced within 3 and 8 h, respectively, VITEK 2 appears a fast and reliable method for detection of GRE in microbiology laboratories.     

Induction of Staphylococcus aureus-specific IgA and agglutination potency in milk of cows by mucosal immunization

Lactating cows were immunized with inactivated Staphylococcus aureus strains and concentrated culture supernatants. Application of a repeated mucosal immunization scheme resulted in significant levels of S. aureus-specific IgA in milk of dairy cows. Average IgA titers against whole cell S. aureus increased during the first 10 weeks of immunization after which a plateau level was reached and maintained during lactation. Immune whey agglutinated both bovine and human S. aureus strains including methicillin-resistant S. aureus (MRSA) strains and recognized extracted S. aureus proteins on Western blot. ELISAs to quantify milk IgA reactive with a number of S. aureus virulence proteins (e.g. enterotoxins, microbial surface component recognizing adhesive matrix molecules (MSCRAMMs) and immune modulating proteins) and cell wall components, demonstrated the polyclonality of the IgA. Correlations observed between agglutination and specific IgA titers for whey and for purified IgA suggested functionality of the induced antibodies. Milk from immunized cows may provide a way of producing potentially therapeutic polyclonal antibodies against S. aureus colonization and infection.     

Faecal carriage of extended‐spectrum β‐lactamase‐producing Enterobacteriaceae among humans in Java,Indonesia, in 2001–2002

Objective To characterise commensal Escherichia coli and other Enterobacteriaceae with reduced susceptibility to cefotaxime that were collected in a large survey carried out among 3995 patients and healthy persons in two urban regions on Java, Indonesia, in 2001–2002. Methods The putative extended‐spectrum β‐lactamase (ESBL)‐producing Enterobacteriaceae were analysed using double‐disk synergy tests, isoelectric focusing, PCR assays, DNA sequencing, and pulsed‐field gel electrophoresis (PFGE). Results On the day of discharge after five or more days of hospitalisation, at least 95 of 999 (9.5%) patients carried ESBL‐positive Enterobacteriaceae as dominant faecal flora. Six patients were simultaneously colonised with E. coli and Klebsiella pneumoniae isolates with ESBL activity. On admission, only 6 of 998 (0.6%) patients were colonised. Faecal carriage of ESBL‐producing Enterobacteriaceae among healthy persons or persons visiting a public health centre was not detected. The 107 ESBL‐positive strains included 68 E. coli, 35 K. pneumoniae, and four other Enterobacteriaceae. bla_CTX‐M‐15 was the most prevalent ESBL in both E. coli (47.1%) and K. pneumoniae (45.7%), but the E. coli O25b‐ST131 clone was virtually absent. Other ESBL types found were: SHV‐2, ‐2a, ‐5, ‐12, CTX‐M‐3, ‐9, ‐14, and TEM‐19. PFGE revealed extensive genetic diversity among the isolates. Conclusions In 2001–2002, faecal carriage of ESBL‐producing Enterobacteriaceae as dominant flora in Indonesia was almost exclusively hospital‐associated. The presence of various bla_ESBL genes and the extensive genetic diversity among isolates argue against a single/dominant strain outbreak.     

Microbial Typing by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry: Do We Need Guidance for Data Interpretation?

The integration of matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) in clinical microbiology has revolutionized species identification of bacteria, yeasts, and molds. However, beyond straightforward identification, the method has also been suggested to have the potential for subspecies-level or even type-level epidemiological analyses. This minireview explores MALDI-TOF MS-based typing, which has already been performed on many clinically relevant species. We discuss the limits of the method''s resolution and we suggest interpretative criteria allowing valid comparison of strain-specific data. We conclude that guidelines for MALDI-TOF MS-based typing can be developed along the same lines as those used for the interpretation of data from pulsed-field gel electrophoresis (PFGE).     

Prevalence of ST9 Methicillin-Resistant Staphylococcus aureus among Pigs and Pig Handlers in Malaysia

Methicillin-resistant Staphylococcus aureus (MRSA) of sequence type 398 (ST398) has frequently been detected in pigs and pig handlers. However, in Malaysia, sampling 360 pigs and 90 pig handlers from 30 farms identified novel ST9-spa type t4358-staphylococcal cassette chromosome mec type V MRSA strains that were found to transiently colonize more than 1% of pigs and 5.5% of pig handlers.Methicillin-resistant Staphylococcus aureus (MRSA) primarily causes human diseases and has recently been identified in pigs and pig handlers. This raised concerns about the role of this porcine reservoir in human infections. In Malaysia, several studies reported the prevalence and characteristics of MRSA isolates from clinical and community settings (15, 20). However, no data have yet been presented on MRSA in pigs. Here, we determined the prevalence of MRSA colonization in pigs and pig handlers in Malaysia.Thirty randomly selected farms in the district of Kuala Langat from the Selangor state of Malaysia were sampled for MRSA. All farms were within a 5-km radius. The farm sizes ranged from 2,000 to 10,000 pigs. Twelve samples (three each from four different age groups, sows, piglets, weanlings, and grower-finishers) were taken at each farm. Nares of the pigs were swabbed by a trained veterinarian. In addition, the three pig handlers per farm (the maximum number of workers in a farm varied from six to eight) who had the highest level of exposure to pigs provided nasal swabs. Employees filled out a questionnaire regarding possible risk factors for MRSA colonization (11). Risk factors included the number of years of work with pigs on that farm, number of hours working with pigs per week, contact with other animals, recent hospitalization, recent treatment with antibiotics, personal or familial skin and soft tissue infection in the last 3 months, and participation in team sports.MRSA was isolated according to methods described previously (13). The antibiotic susceptibility of the strains against the antibiotics mentioned below was tested by using the Kirby-Bauer disk diffusion method. The results of susceptibility testing were interpreted according to accepted guidelines (4). MRSA isolates were PCR tested for the mecA gene and subjected to staphylococcal protein A gene (spa) sequencing (http://spaserver.ridom.de), staphylococcal cassette chromosome mec (SCCmec) typing (22), and multilocus sequence typing (http://www.mlst.net). All isolates were screened for the virulence genes pvl (14), fnb and cna (1), sea, seb, sec, sed, see, seg, tsst, eta, and etb (8).MRSA was isolated from one or more pigs on 30% (9/30) of the farms. The overall prevalence of MRSA among pigs was found to be 1.38% (5/360), with 5.3% (4/75) in weanlings and 1.3% (1/75) in grower-finishers. None of the piglets or sows was colonized. Except for farm 1, in all other eight farms, only one animal was colonized. The prevalence of MRSA colonization in humans was 5.5% (5/90). None of the risk factors identified in the questionnaire were found to have a significant association with human colonization. MRSA was not isolated from both pigs and pig handlers on any of the farms. When MRSA-positive animals were tested a second time, no MRSA was isolated. Unfortunately, no secondary samples were taken from humans. This indicates that MRSA is only transiently present, similar to what we found in a study with healthy Malaysians (16).Susceptibility testing revealed 100% resistance to erythromycin, ceftriaxone, cefoxitin, ciprofloxacin, gentamicin, tetracycline, trimethoprim-sulfamethoxazole, clindamycin, and quinupristin-dalfopristine, while tigecycline, cephalexin (cefalexin), and fusidic acid showed 80%, 70%, and 20% resistance, respectively. No resistance was observed for mupirocin, amikacin, linezolid, vancomycin, and netilmicin. The surprising pattern of resistance to clindamycin, quinupristin-dalfopristine, and tigecycline was confirmed after three repeats of the test.Molecular typing showed that MRSA isolates belonged to two sequence types: ST9 (spa type t4358) and ST1 (spa type t1784). Except for strains from handlers on farm 12 and 29 (ST1), all other isolates were ST9 (Table ​(Table1).1). All isolates carried SCCmec V. Virulence gene analysis revealed enterotoxin genes, such as seb (60%), see (10%), and seg (90%), and microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) that include Cna (20%, only in ST1 isolates) and Fnb (100%). None of the MRSA isolates carried pvl, eta, etb, tsst, or enterotoxin genes other than seb, see, and seg (Table ​(Table11).

TABLE 1.

Characteristics of MRSA isolates from pigs and pig handlers in Malaysia

Distribution of the serine-aspartate repeat protein-encoding sdr genes among nasal-carriage and invasive Staphylococcus aureus strains

The sdr locus was found in all 497 investigated Staphylococcus aureus strains, although in 29 strains it contained only the sdrC gene (sdrD negative, sdrE negative). The sdrC-positive, sdrD-negative, sdrE-negative gene profile was exclusive to methicillin-sensitive S. aureus (MSSA) strains (Fisher's exact test; P = 0.0005) and was not found in the strains collected from bone infections (P = 0.0019). We also found a strong association between the presence of the sdrD gene and methicillin-resistant S. aureus strains (P < 0.0001). Our findings suggest that MSSA strains with the newly uncovered sdrC-positive, sdrD-negative, sdrE-negative gene profile have a substantially decreased potential to establish bone infection.     

Natural antibodies against several pneumococcal virulence proteins in children during the pre-pneumococcal-vaccine era: the generation R study

The currently available pneumococcal vaccines do not protect against all serotypes of Streptococcus pneumoniae. A shift toward nonvaccine serotypes causing colonization and invasive disease has occurred, and studies on protein-based vaccines have been undertaken. We assessed the association between specific antibodies against pneumococcal virulence proteins and colonization and respiratory tract infections (RTIs). Additionally, we assessed the extent to which colonization induces a humoral immune response. Nasopharyngeal swabs collected from children at 1.5, 6, 14, and 24 months of age were cultured for pneumococcus. Serum samples were obtained at birth and at 6, 14, and 24 months (n = 57 children providing 177 serum samples). Data were collected prior to the pneumococcal vaccine era. IgG, IgA, and IgM levels against 17 pneumococcal protein vaccine candidates were measured using a bead-based flow cytometry technique (xMAP; Luminex Corporation). Information regarding RTIs was questionnaire derived. Levels of IgG against all proteins were high in cord blood, decreased in the first 6 months and increased again thereafter, in contrast to the course of IgA and IgM levels. Specific antibodies were induced upon colonization. Increased levels of IgG against BVH-3, NanA, and SP1003 at 6 months, NanA, PpmA, PsaA, SlrA, SP0189, and SP1003 at 14 months, and SlrA at 24 months were associated with a decreased number of RTIs in the third year of life but not with colonization. Maternal antipneumococcal antibodies did not protect against pneumococcal colonization and infection. Certain antibodies against pneumococcal virulence proteins, some of which are induced by colonization, are associated with a decreased number of RTIs in children. This should be taken into account in future pneumococcal vaccine studies.