Antimicrobial Susceptibility of Invasive Streptococcus pyogenes Isolates in Germany during 2003-2013

A nationwide laboratory-based surveillance study of invasive S. pyogenes infections was conducted in Germany. Invasive isolates (n = 1,281) were obtained between 2003 and 2013. All isolates were susceptible to penicillin, cefotaxime and vancomycin. Tetracycline showed the highest rate of resistant or intermediate resistant isolates with 9.8%, followed by macrolides (4.0%), trimethoprim/sulfamethoxazole (SXT) (1.9%), levofloxacin (1.3%), chloramphenicol (0.9%) and clindamycin (0.7%). The most prominent trends were the appearance of levofloxacin non-susceptible isolates since 2011, and an increase of SXT non-susceptibility since 2012.     

Disease Burden of Invasive Listeriosis and Molecular Characterization of Clinical Isolates in Taiwan, 2000-2013

The information about disease burden and epidemiology of invasive listeriosis in Asia is scarce. From 2000 to 2013, a total of 338 patients with invasive listeriosis (bacteremia, meningitis, and peritonitis) were treated at four medical centers in Taiwan. The incidence (per 10,000 admissions) of invasive listeriosis increased significantly during the 14-year period among the four centers (0.15 in 2000 and >1.25 during 2010–2012) and at each of the four medical centers. Among these patients, 45.9% were elderly (>65 years old) and 3.3% were less than one year of age. More than one-third (36.7%) of the patients acquired invasive listeriosis in the spring (April to June). Among the 132 preserved Listeria monocytogenes isolates analyzed, the most frequently isolated PCR serogroup-sequence type (ST) was IIb-ST87 (23.5%), followed by IIa-ST378 (19.7%) and IIa-ST155 (12.1%). Isolation of PCR serogroups IIb and IVb increased significantly with year, with a predominance of IIb-ST87 isolates (23.5%) and IIb-ST 228 isolates emerging in 2013. A total of 12 different randomly amplified polymorphic DNA (RAPD) patterns (Patterns I to XII) were identified among the 112 L. monocytogenes isolates belonging to eight main PCR serogroup-STs. Identical RAPD patterns were found among the isolates exhibiting the same PCR serogroup-ST. In conclusion, our study revealed that during 2000–2013, listeriosis at four medical centers in Taiwan was caused by heterogeneous strains and that the upsurge in incidence beginning in 2005 was caused by at least two predominant clones.     

Characterization of Streptococcus suis Isolates from Slaughter Swine

The characterization of 61 Streptococcus suis strains isolated from Chinese slaughter pigs was investigated. S. suis serotypes 1, 2, 3, 4, 7, 9, 10, 11, 12, 13, 14, 15, 16, 22, 23, 25, 26, 28, 29, and 1/2 were found in the isolates by serum agglutination. Of all the prevalent serotypes, S. suis serotype 7 is the most predominant circulating in Chinese slaughter pigs. The virulence-associated genes profile and multilocus sequence typing scheme of the isolates were analyzed. The mrp-/epf-/sly- virulence-associated genes type is the most prevalent in the isolates from slaughter pigs. It is the first time to find S. suis serotypes 7 and 9 isolates with epf. The serotypes 7 and 9 isolates with mrp and/or epf genes did not express MRP and/or EF in the present research. Thirteen new ST types were identified for the first time. ST1 complex and ST27 complex of S. suis are prevalent in China. This paper supplied information to understand the characteristics, such as capsular serotypes, virulence factors, and gene backgrounds of S. suis carried by slaughter pigs.     

Zn2+ Uptake in Streptococcus pyogenes: Characterization of adcA and lmb Null Mutants

An effective regulation of metal ion homeostasis is essential for the growth of microorganisms in any environment and in pathogenic bacteria is strongly associated with their ability to invade and colonise their hosts. To gain a better insight into zinc acquisition in Group A Streptococcus (GAS) we characterized null deletion mutants of the adcA and lmb genes of Streptococcus pyogenes strain MGAS5005 encoding the orthologues of AdcA and AdcAII, the two surface lipoproteins with partly redundant roles in zinc homeostasis in Streptococcus pneumoniae. Null adcA and lmb mutants were analysed for their capability to grow in zinc-depleted conditions and were found to be more susceptible to zinc starvation, a phenotype that could be rescued by the addition of Zn²⁺ ions to the growth medium. Expression of AdcA, Lmb and HtpA, the polyhistidine triad protein encoded by the gene adjacent to lmb, during growth under conditions of limited zinc availability was examined by Western blot analysis in wild type and null mutant strains. In the wild type strain, AdcA was always present with little variation in expression levels between conditions of excess or limited zinc availability. In contrast, Lmb and HtpA were expressed at detectable levels only during growth in the presence of low zinc concentrations or in the null adcA mutant, when expression of lmb is required to compensate for the lack of adcA expression. In the latter case, Lmb and HtpA were overexpressed by several fold, thus indicating that also in GAS AdcA is a zinc-specific importer and, although it shares this function with Lmb, the two substrate-binding proteins do not show fully overlapping roles in zinc homeostasis.     

Morphological and Molecular Characterization of Fusarium solani Isolates

Fusarium solani is a species complex (FSSC) containing isolates that cause diseases in important crops such as root and fruit rot of Cucurbita spp., root and stem rot of pea, sudden death syndrome of soybean, foot rot of bean and dry rot of potato tubers during storage. Based on host range tests, F. solani were subdivided into different formae specialis (f. sp.) and varieties, while DNA sequences of 28S rDNA, internally transcribed spacers (ITS) rDNA and elongation factor (EF-1α) distinguished the ' F. solani complex' in 50 subspecific lineages. In this study we characterized, by cultural, morphological and molecular criteria, 34 isolates of F. solani obtained from potato, other crops and soil. The 34 isolates in the FSSC showed wide variability for their cultural, morphological and molecular traits. The wide variability observed with amplified fragment-length polymorphism (AFLP) and mini-microsatellite analyses is in agreement with the polymorphism observed, in a previous study, within FSSC. Nine of 34 isolates in the FSSC, classified as F. solani var. coeruleum , were morphologically distinguishable from the other F. solani isolates but they were distributed in different clusters; moreover, the nine isolates showed instability of the coeruleum pigmentation of the colonies, supporting the ambiguity of the taxa of this variety of F. solani. Using sequence data from ITS plus 5.8S rDNA region, the isolates were classified into different clades. In particular eight isolates were classified into a well-supported clade including F. solani f. sp . pisi , nine into a clade including only isolates of F. solani f. sp . radicicola and four into a clade including F. solani f. sp . cucurbitae , but this classification could not be used if is not in agreement with host specificity. Two of the nine F. solani var. coeruleum isolates were phylogenetically distinct from all the other FSSC strains.     

Variation in sic Gene Encoding Complement-Inhibiting Protein of Streptococcus pyogenes Serotype M1 Isolates in Japan

DNA sequencing of the gene encoding a complement-inhibiting protein of Streptococcus pyogenes (streptococcal inhibitor of complement, Sic) was carried out on 49 strains of S. pyogenes serotype M1. Those strains were obtained from patients and asymptomatic carriers in Japan from 1969 to 1997 and had various pulsed-field gel electrophoresis (PFGE) patterns. Four identical polymorphic sites were found in the strains with the same PFGE pattern (Ia), but not in those giving the pattern IIa. The other identical sites were found in the strains with the PFGE pattern IIa, but not in those with the pattern Ia. These observations suggest that each of PFGE patterns was restricted to a set of variation in the sic gene. Received: 13 January 2000 / Accepted: 24 February 2000     

Molecular anatomy of the Streptococcus pyogenes pSM19035 partition and segrosome complexes

Vancomycin or erythromycin resistance and the stability determinants, δω and ωεζ, of Enterococci and Streptococci plasmids are genetically linked. To unravel the mechanisms that promoted the stable persistence of resistance determinants, the early stages of Streptococcus pyogenes pSM19035 partitioning were biochemically dissected. First, the homodimeric centromere-binding protein, ω2, bound parS DNA to form a short-lived partition complex 1 (PC1). The interaction of PC1 with homodimeric δ [δ2 even in the apo form (Apo-δ2)], significantly stimulated the formation of a long-lived ω2·parS complex (PC2) without spreading into neighbouring DNA sequences. In the ATP·Mg2+ bound form, δ2 bound DNA, without sequence specificity, to form a transient dynamic complex (DC). Second, parS bound ω2 interacted with and promoted δ2 redistribution to co-localize with the PC2, leading to transient segrosome complex (SC, parS·ω2·δ2) formation. Third, δ2, in the SC, interacted with a second SC and promoted formation of a bridging complex (BC). Finally, increasing ω2 concentrations stimulated the ATPase activity of δ2 and the BC was disassembled. We propose that PC, DC, SC and BC formation were dynamic processes and that the molar ω2:δ2 ratio and parS DNA control their temporal and spatial assembly during partition of pSM19035 before cell division.     

Epidemiology of Streptococcus pneumoniae Serogroup 6 Isolates from IPD in Children and Adults in Germany

This study presents serogroup 6 isolates from invasive pneumococcal disease (IPD) before and after the recommendation for childhood pneumococcal conjugate vaccination in Germany (July 2006). A total of 19,299 (children: 3508, adults: 15,791) isolates were serotyped. Serogroup 6 isolates accounted for 9.5% (children) and 6.7% (adults), respectively. 548 isolates had serotype 6A, 558 had serotype 6B, 285 had serotype 6C, and 4 had serotype 6D. Among children, serotype 6B was most prevalent (7.5% of isolates) before vaccination, followed by 6A and 6C. After the 7-valent pneumococcal conjugate vaccine (PCV7), the prevalence of serotype 6B significantly decreased (p = 0.040), a pattern which continued in the higher-valent PCV period (PCV10, PCV13). Serotype 6A prevalence showed a slight increase directly after the start of PCV7 vaccination, followed by a decrease which continued throughout the PCV10/13 period. Serotype 6C prevalence remained low. Serotype 6D was not found among IPD isolates from children. Among adults, prevalence of both 6A and 6B decreased, with 6B reaching statistical significance (p = 0.045) and 6A showing a small increase in 2011–2012. Serotype 6C prevalence was 1.5% or lower before vaccination, but increased post-vaccination to 3.6% in 2011/12 (p = 0.031). Four serotype 6D isolates were found post-PCV7 childhood vaccination, and two post-PCV10/13. Antibiotic resistance was found mainly in serotype 6B; serotype 6A showed lower resistance rates. Serotype 6C isolates only showed resistance among adults; serotype 6D isolates showed no resistance. Multilocus sequence typing showed that sequence type (ST) 1692 was the most prevalent serotype 6C clone. Thirty-two other STs were found among serotype 6C isolates, of which 12 have not been previously reported. The four serotype 6D isolates had ST 948, ST 2185 and two new STs: 8422 and 8442. Two serogroup 6 isolates could not be assigned to a serotype, but had STs common to serogroup 6.     

Molecular Epidemiology of Nontypeable Haemophilus influenzae Causing Community-Acquired Pneumonia in Adults

Nontypeable Haemophilus influenzae (NTHi) is an opportunistic pathogen which causes a variety of respiratory infections. The objectives of the study were to determine its antimicrobial susceptibility, to characterize the β-lactam resistance, and to establish a genetic characterization of NTHi isolates. Ninety-five NTHi isolates were analyzed by pulsed field gel electrophoresis (PFGE) and multi locus sequence typing (MLST). Antimicrobial susceptibility was determined by microdilution, and the ftsI gene (encoding penicillin-binding protein 3, PBP3) was PCR amplified and sequenced. Thirty (31.6%) isolates were non-susceptible to ampicillin (MIC≥2 mg/L), with 10 of them producing β-lactamase type TEM-1 as a resistance mechanism. After ftsI sequencing, 39 (41.1%) isolates showed amino acid substitutions in PBP3, with Asn526→ Lys being the most common (69.2%). Eighty-four patients were successfully treated with amoxicillin/clavulanic acid, ceftriaxone and levofloxacin. Eight patients died due either to aspiration or complication of their comorbidities. In conclusion, NTHi causing CAP in adults shows high genetic diversity and is associated with a high rate of reduced susceptibility to ampicillin due to alterations in PBP3. The analysis of treatment and outcomes demonstrated that NTHi strains with mutations in the ftsI gene could be successfully treated with ceftriaxone or fluoroquinolones.     

Molecular Characterization of Cryptosporidium Isolates from Humans and Animals in Iran

Isolates of Cryptosporidium spp. from human and animal hosts in Iran were characterized on the basis of both the 18S rRNA gene and the Laxer locus. Three Cryptosporidium species, C. hominis, C. parvum, and C. meleagridis, were recognized, and zoonotically transmitted C. parvum was the predominant species found in humans.     

Microbiological and Molecular Characterization of Staphylococcus hominis Isolates from Blood

Background

Among Coagulase-Negative Staphylococci (CoNS), Staphylococcus hominis represents the third most common organism recoverable from the blood of immunocompromised patients. The aim of this study was to characterize biofilm formation, antibiotic resistance, define the SCCmec (Staphylococcal Chromosomal Cassette mec) type, and genetic relatedness of clinical S. hominis isolates.

Methodology

S. hominis blood isolates (n = 21) were screened for biofilm formation using crystal violet staining. Methicillin resistance was evaluated using the cefoxitin disk test and the mecA gene was detected by PCR. Antibiotic resistance was determined by the broth microdilution method. Genetic relatedness was determined by pulsed-field gel electrophoresis (PFGE) and SCCmec typed by multiplex PCR using two different methodologies described for Staphylococcus aureus.

Results

Of the S. hominis isolates screened, 47.6% (10/21) were categorized as strong biofilm producers and 23.8% (5/21) as weak producers. Furthermore, 81% (17/21) of the isolates were methicillin resistant and mecA gene carriers. Resistance to ampicillin, erythromycin, and trimethoprim was observed in >70% of isolates screened. Each isolate showed a different PFGE macrorestriction pattern with similarity ranging between 0–95%. Among mecA-positive isolates, 14 (82%) harbored a non-typeable SCCmec type: eight isolates were not positive for any ccr complex; four contained the mec complex A ccrAB1 and ccrC, one isolate contained mec complex A, ccrAB4 and ccrC, and one isolate contained the mec complex A, ccrAB1, ccrAB4, and ccrC. Two isolates harbored the association: mec complex A and ccrAB1. Only one strain was typeable as SCCmec III.

Conclusions

The S. hominis isolates analyzed were variable biofilm producers had a high prevalence of methicillin resistance and resistance to other antibiotics, and high genetic diversity. The results of this study strongly suggested that S. hominis isolates harbor new SCCmec structural elements and might be reservoirs of ccrC1 in addition to ccrAB1 and mec complex A.     

Molecular Characterization of Pneumococcal Isolates from Pets and Laboratory Animals

Background

Between 1986 and 2008 Streptococcus pneumoniae was isolated from 41 pets/zoo animals (guinea pigs (n = 17), cats (n = 12), horses (n = 4), dogs (n = 3), dolphins (n = 2), rat (n = 2), gorilla (n = 1)) treated in medical veterinary laboratories and zoos, and 44 laboratory animals (mastomys (multimammate mice; n = 32), mice (n = 6), rats (n = 4), guinea pigs (n = 2)) during routine health monitoring in an animal facility. S. pneumoniae was isolated from nose, lung and respiratory tract, eye, ear and other sites.

Methodology/Principal Findings

Carriage of the same isolate of S. pneumoniae over a period of up to 22 weeks was shown for four mastomys. Forty-one animals showed disease symptoms. Pneumococcal isolates were characterized by optochin sensitivity, bile solubility, DNA hybridization, pneumolysin PCR, serotyping and multilocus sequence typing. Eighteen of the 32 mastomys isolates (56%) were optochin resistant, all other isolates were optochin susceptible. All mastomys isolates were serotype 14, all guinea pig isolates serotype 19F, all horse isolates serotype 3. Rats had serotypes 14 or 19A, mice 33A or 33F. Dolphins had serotype 23F, the gorilla serotype 14. Cats and dogs had many different serotypes. Four isolates were resistant to macrolides, three isolates also to clindamycin and tetracyclin. Mastomys isolates were sequence type (ST) 15 (serotype 14), an ST/serotype combination commonly found in human isolates. Cats, dogs, pet rats, gorilla and dolphins showed various human ST/serotype combinations. Lab rats and lab mice showed single locus variants (SLV) of human STs, in human ST/serotype combinations. All guinea pig isolates showed the same completely new combination of known alleles. The horse isolates showed an unknown allele combination and three new alleles.

Conclusions/Significance

The isolates found in mastomys, mice, rats, cats, dogs, gorilla and dolphins are most likely identical to human pneumococcal isolates. Isolates from guinea pigs and horses appear to be specialized clones for these animals. Our data redraw attention to the fact that pneumococci are not strictly human pathogens. Pet animals that live in close contact to humans, especially children, can be infected by human isolates and also carriage of even resistant isolates is a realistic possibility.     

Morphological and Molecular Characterization of Meloidogyne mayaguensis Isolates from Florida

The discovery of Meloidogyne mayaguensis is confirmed in Florida; this is the first report for the continental United States. Meloidogyne mayaguensis is a virulent species that can reproduce on host cultivars bred for nematode resistance. The perineal patterns of M. mayaguensis isolates from Florida show morphological variability and often are similar to M. incognita. Useful morphological characters for the separation of M. mayaguensis from M. incognita from Florida are the male stylet length values (smaller for M. mayaguensis than M. incognita) and J2 tail length values (greater for M. mayaguensis than M. incognita). Meloidogyne mayaguensis values for these characters overlap with those of M. arenaria and M. javanica from Florida. Enzyme analyses of Florida M. mayaguensis isolates show two major bands (VS1-S1 phenotype) of esterase activity, and one strong malate dehydrogenase band (Rm 1.4) plus two additional weak bands that migrated close together. Their detection requires larger amounts of homogenates from several females. Amplification of two separate regions of mitochondrial DNA resulted in products of a unique size. PCR primers embedded in the COII and 16S genes produced a product size of 705 bp, and amplification of the 63-bp repeat region resulted in a single product of 322 bp. Nucleotide sequence comparison of these mitochondrial products together with sequence from 18S rDNA and ITS1 from the nuclear genome were nearly identical with the corresponding regions from a M. mayaguensis isolate from Mayaguez, Puerto Rico, the type locality of the species. Meloidogyne mayaguensis reproduced on cotton, pepper, tobacco, and watermelon but not on peanut. Preliminary results indicate the M. mayaguensis isolates from Florida can reproduce on tomato containing the Mi gene. Molecular techniques for the identification of M. mayaguensis will be particularly useful in cases of M. mayaguensis populations mixed with M. arenaria, M. incognita, and M. javanica, which are the most economically important root-knot nematode species in Florida, and especially when low (<25) numbers of specimens of these species are recovered from the soil.     

Molecular Characterization and Antimicrobial Susceptibility of Staphylococcus aureus Isolates from Clinical Infection and Asymptomatic Carriers in Southwest Nigeria

Few reports from Africa suggest that resistance pattern, virulence factors and genotypes differ between Staphylococcus aureus from nasal carriage and clinical infection. We therefore compared antimicrobial resistance, selected virulence factors and genotypes of S. aureus from nasal carriage and clinical infection in Southwest Nigeria. Non-duplicate S. aureus isolates were obtained from infection (n = 217) and asymptomatic carriers (n = 73) during a cross sectional study in Lagos and Ogun States, Nigeria from 2010–2011. Susceptibility testing was performed using Vitek automated systems. Selected virulence factors were detected by PCR. The population structure was assessed using spa typing. The spa clonal complexes (spa-CC) were deduced using the Based Upon Repeat Pattern algorithm (BURP). Resistance was higher for aminoglycosides in clinical isolates while resistances to quinolones and tetracycline were more prevalent in carrier isolates. The Panton-Valentine leukocidin (PVL) was more frequently detected in isolates from infection compared to carriage (80.2 vs 53.4%; p<0.001, chi²-test). Seven methicillin resistant S. aureus isolates were associated with spa types t002, t008, t064, t194, t8439, t8440 and t8441. The predominant spa types among the methicillin-susceptible S. aureus isolates were t084 (65.5%), t2304 (4.4%) and t8435 (4.1%). spa-CC 084 was predominant among isolates from infection (80.3%, n = 167) and was significantly associated with PVL (OR = 7.1, 95%CI: 3.9–13.2, p<0.001, chi^2- test). In conclusion, PVL positive isolates were more frequently detected among isolates from infection compared to carriage and are associated with spa-CC 084.