Characterization of sil in invasive group A and G streptococci: antibodies against bacterial pheromone peptide SilCR result in severe infection.

Group G beta-hemolytic streptococcus (GGS) strains cause severe invasive infections, mostly in patients with comorbidities. GGS is known to possess virulence factors similar to those of its more virulent counterpart group A streptococcus (GAS). A streptococcal invasion locus, sil, was identified in GAS. sil encodes a competence-stimulating peptide named SilCR that activates bacterial quorum sensing and has the ability to attenuate virulence in GAS infections. We found that sil is present in most GGS strains (82%) but in only 25% of GAS strains, with a similar gene arrangement. GGS strains that contained sil expressed the SilCR peptide and secreted it into the growth medium. In a modified murine model of GGS soft tissue infection, GGS grown in the presence of SilCR caused a milder disease than GGS grown in the absence of SilCR. To further study the role of the peptide in bacterial virulence attenuation, we vaccinated mice with SilCR to produce specific anti-SilCR antibodies. Vaccinated mice developed a significantly more severe illness than nonvaccinated mice. Our results indicate that the sil locus is much more prevalent among the less virulent GGS strains than among GAS strains. GGS strains express and secrete SilCR, which has a role in attenuation of virulence in a murine model. We show that the SilCR peptide can protect mice from infection caused by GGS. Furthermore, vaccinated mice that produce specific anti-SilCR antibodies develop a significantly more severe infection. To our knowledge, this is a novel report demonstrating that specific antibodies against a bacterial component cause more severe infection by those bacteria.

KPC-9, a novel carbapenemase from clinical specimens in Israel.

A bla(KPC-9) carbapenemase variant was discovered in isolates of Klebsiella pneumoniae and Escherichia coli from a single patient. It differed from bla(KPC-3) by one amino acid substitution (Val239Ala). The K. pneumoniae isolate was typed as ST258, as was the epidemic Israeli KPC-3 clone. bla(KPC-9) was found on a plasmid indistinguishable from pKpQIL that carries bla(KPC-3) in the epidemic clone. Compared to KPC-3, KPC-9 conferred less resistance to carbapenems and higher resistance to ceftazidime.

Comparison of two carbapenem-resistant Klebsiella pneumoniae clones: from a contained outbreak in a paediatric population and from a national epidemic.

OBJECTIVES: A refractory epidemic of carbapenem-resistant Klebsiella pneumoniae (CRKP) emerged in the adult population at our hospital in 2005, as in most Israeli hospitals. Contemporaneously, a different clone of CRKP caused an easily contained outbreak in a paediatric long-term care facility (LTCF) in Jerusalem. While previously identified host-related risk factors for colonization by these organisms undoubtedly contributed to these outbreaks, it is very likely that bacterial factors might be crucial in explaining the striking differences in transmissibility between the implicated strains. We therefore sought bacterial factors associated with these different epidemiological behaviours. METHODS: Seven CRKP isolated at our hospital and the LTCF during 2008-09 were examined by antimicrobial susceptibility testing and PFGE, and further analyses of these two clones was done using multilocus sequence typing and competition experiments. Plasmids were analysed by conjugation, restriction mapping, PCR and sequencing. RESULTS: Both clones were multidrug resistant and harboured identical plasmids carrying the bla(KPC-3) gene. The hyper-transmissible epidemic clone carried additional antibiotic resistance genes and hosted an additional plasmid. The clone from the LTCF did not demonstrate hyper-transmissible properties despite its presence in an institution of a type commonly plagued by the epidemic clone. Competition assays showed the more easily contained strain to be fitter. CONCLUSIONS: These findings suggest that neither the presence of the plasmid carrying the bla(KPC-3) gene nor relative survival fitness account for the hyper-transmissibility of the epidemic strain. The role of patient age in susceptibility to colonization by the epidemic strain should be investigated.

A carbapenem-resistant Klebsiella pneumoniae epidemic clone in Jerusalem: sequence type 512 carrying a plasmid encoding aac(6')-Ib.

OBJECTIVES: We characterized distinctive features of a hypertransmissible carbapenem-resistant Klebsiella pneumoniae (CRKP) clone that emerged at Hadassah Hospital, Ein-Kerem, Jerusalem, Israel, in 2006. METHODS: Eleven CRKP isolated at Hadassah Hospital during 2005-09 were examined by antimicrobial susceptibility testing, PFGE and multilocus sequence typing (MLST). Plasmids were analysed by conjugation, restriction mapping, PCR and sequencing. RESULTS: Divergence from the national epidemic sequence type (ST) ST258 to ST512 was observed early on. Carbapenem resistance was conferred by bla(KPC-3) carried on a plasmid apparently closely related to pKpQIL, also from Israel. This clone also carried a 15 kb plasmid, designated pAAC154, that carries a Tn1331 derivative containing the aac(6')-Ib gene. pAAC154 does not carry a bla(KPC) gene, but is similar to pS15, a plasmid from New York that carries bla(KPC-2). CONCLUSIONS: A single CRKP clone ST512 has spread efficiently in our region. In this clone, aac(6')-Ib, common in CRKP strains, is carried on a different plasmid from bla(KPC-3).

Macrolide resistance in Mycoplasma pneumoniae, Israel, 2010.

Macrolide resistance in Mycoplasma pneumoniae is often found in Asia but is rare elsewhere. We report the emergence of macrolide-resistant M. pneumoniae in Israel and the in vivo evolution of such resistance during the treatment of a 6-year-old boy with pneumonia.

Imipenem disc for detection of KPC carbapenemase-producing Enterobacteriaceae in clinical practice.

The global spread of class A-carbapenemase-producing Enterobacteriaceae has made the development of a simple test a desirable goal. A disc diffusion test using imipenem was 100% sensitive and 96% specific in identifying carbapenemase-producing organisms, potentially reducing or eliminating the need for the relatively labor-intensive modified Hodge test.

An outbreak of achromobacter xylosoxidans associated with ultrasound gel used during transrectal ultrasound guided prostate biopsy.

PURPOSE: We describe an outbreak of Achromobacter xylosoxidans after transrectal ultrasound guided prostate biopsy at a urology unit at a tertiary care center as well as clinical and microbiological investigation, and intervention. MATERIALS AND METHODS: In September 2008, several days after undergoing transrectal ultrasound guided prostate biopsy, 4 patients were hospitalized with fever. We reviewed the procedure and infection control practices in the urology service. Environmental cultures were obtained from equipment and materials used for the procedure. Isolates were identified by routine laboratory procedures with molecular confirmation and characterized by pulsed field gel electrophoresis. RESULTS: A. xylosoxidans was isolated from the urine of 2 patients, of whom 1 also had a positive blood culture. Review of transrectal ultrasound guided prostate biopsy revealed that the lubricant gel used in the procedure, which the biopsy needle passes through, was held in a plastic container that was repeatedly refilled from a large bag. A. xylosoxidans was isolated from this container. Pulsed field gel electrophoresis showed that the isolates obtained from patients and the gel were identical. CONCLUSIONS: Contaminated lubricant gel was the cause of this outbreak. The practice of repeatedly refilling gel containers with nonsterile gel was replaced by the use of individual sterile gel sachets in each patient. No further cases occurred. During an invasive procedure involving a sterile body site, such as transrectal ultrasound guided prostate biopsy, using sterile gel is essential. Our experience emphasizes the crucial need to review all invasive procedures from an infection control perspective.

High-resolution melt curve analysis for identification of single nucleotide mutations in the quinolone resistance gene aac(6')-Ib-cr.

We have developed a simple PCR-based high-resolution melt curve analysis for identification of the quinolone resistance gene aac(6')-Ib-cr through regions encompassing the two defining single nucleotide mutations. Dissociation curves showed 100% concordance with DNA sequencing, including the identification of a strain where aac(6')-Ib and aac(6')-Ib-cr coexist.

Imported melioidosis, Israel, 2008.

In 2008, melioidosis was diagnosed in an agricultural worker from Thailand in the southern Jordan Valley in Israel. He had newly diagnosed diabetes mellitus, fever, multiple abscesses, and osteomyelitis. Burkholderia pseudomallei was isolated from urine and blood. Four of 10 laboratory staff members exposed to the organism received chemoprophylaxis, 3 of whom had adverse events.

Invasive Scytalidium dimidiatum infection in an immunocompetent adult.

Scytalidium dimidiatum, a dematiaceous fungus, has been well established as an agent of dermatomycosis. There are few reports of invasive infection caused by S. dimidiatum; most infections occurred in immunocompromised hosts. We present an immunocompetent patient with pleural S. dimidiatum infection and review nine other published cases of invasive S. dimidiatum infections.

Multi-center evaluation of one commercial and 12 in-house real-time PCR assays for detection of Mycoplasma pneumoniae.

Detection of Mycoplasma pneumoniae by real-time PCR is not yet standardized across laboratories. We have implemented a standardization protocol to compare the performance of thirteen commercial and in-house approaches. Despite differences on threshold values of samples, all assays were able to detect at least 20M. pneumoniae genomes per reaction.

Effect of a bacterial pheromone peptide on host chemokine degradation in group A streptococcal necrotising soft-tissue infections.

BACKGROUND: Necrotising soft-tissue infections due to group A streptococcus (GAS) are rare (about 0.2 cases per 100000 people). The disease progresses rapidly, causing severe necrosis and hydrolysis of soft tissues. Histopathological analysis of necrotic tissue debrided from two patients (one with necrotising fasciitis and one with myonecrosis) showed large quantities of bacteria but no infiltrating neutrophils. We aimed to investigate whether the poor neutrophil chemotaxis was linked with the ability of group A streptococcus (GAS) to degrade host chemokines. METHODS: We did RT-PCR, ELISA, and dot-blot assays to establish whether GAS induces synthesis of interleukin 8 mRNA, but subsequently degrades the released chemokine protein. Class-specific protease inhibitors were used to characterise the protease that degraded the chemokine. We used a mouse model of human soft-tissue infections to investigate the pathogenic relevance of GAS chemokine degradation, and to test the therapeutic effect of a GAS pheromone peptide (SilCR) that downregulates activity of chemokine protease. FINDINGS: The only isolates from the necrotic tissue were two beta-haemolytic GAS strains of an M14 serotype. A trypsin-like protease released by these strains degraded human interleukin 8 and its mouse homologue MIP2. When innoculated subcutaneously in mice, these strains produced a fatal necrotic soft-tissue infection that had reduced neutrophil recruitment to the site of injection. The M14 GAS strains have a missense mutation in the start codon of silCR, which encodes a predicted 17 aminoacid pheromone peptide, SilCR. Growth of the M14 strain in the presence of SilCR abrogated chemokine proteolysis. When SilCR was injected together with the bacteria, abundant neutrophils were recruited to the site of infection, bacteria were cleared without systemic spread, and the mice survived. The therapeutic effect of SilCR was also obtained in mice challenged with M1 and M3 GAS strains, a leading cause of invasive infections. INTERPRETATION: The unusual reduction in neutrophils in necrotic tissue of people with GAS soft-tissue infections is partly caused by a GAS protease that degrades interleukin 8. In mice, degradation can be controlled by administration of SilCR, which downregulates GAS chemokine protease activity. This downregulation increases neutrophil migration to the site of infection, preventing bacterial spread and development of a fulminant lethal systemic infection.

Streptococcal pyrogenic exotoxin G gene in blood and pharyngeal isolates of Streptococcus dysgalactiae subspecies equisimilis has a limited role in pathogenesis.

BACKGROUND: Streptococcus dysgalactiae subspecies equisimilis (SE) causes human infections that clinically resemble infections due to Streptococcus pyogenes (SP). SE expresses several virulence determinants initially identified in SP, including genes encoding streptococcal pyrogenic exotoxins. SE isolates from patients with toxic shock syndrome were found to harbor a gene designated spegg, which is similar to the SP pyrogenic exotoxin-G gene, termed speG. Other streptococcal pyrogenic exotoxins known to exist in SP were not detected. METHODS: To determine the prevalence of the superantigen gene, spegg, we examined 65 invasive SE from patients presenting from 1989 to 2008 with bacteremia secondary to a variety of illnesses including two patients who fulfilled the criteria for toxic shock syndrome, in comparison with 46 noninvasive pharyngeal isolates. All isolates were tested for the presence of spegg by polymerase chain reaction. Forty-four of the 65 blood isolates were also characterized by emm typing. RESULTS: spegg was identified in 49.2% and 69.5% of the blood and pharyngeal isolates, respectively. emm typing revealed the presence of 13 distinct types. There was no association between clinical presentation and the presence of spegg. We found an association between the presence of spegg and the emm type (p < 0.001). The emm types stG485 and stG840 were more frequent among spegg positive isolates, and stG4222, stG6, and stG166b were associated with spegg negative isolates. CONCLUSION: We found a high prevalence of spegg in invasive and noninvasive SE isolates, associated with specific emm types. Our finding suggests that this gene does not have a role in the pathogenesis of bacteremia.

Bordetella holmesii meningitis in an asplenic patient with systemic lupus erythematosus.

Bordetella holmesii is a slow-growing, Gram-negative, non-oxidizing bacillus with colonies that produce a brown soluble pigment and was originally described by Weyant et al. (1995) as CDC nonoxidizer group 2 (NO-2). It has recently been shown that B. holmesii may be isolated from nasopharyngeal specimens of up to 20% of patients with pertussis-like symptoms. However, invasive B. holmesii has rarely been reported and in the vast majority of cases the patients were immune deficient, mostly as a result of splenectomy or functional asplenia. Clinical presentations have included endocarditis, pneumonia, cellulitis, suppurative arthritis, pyelonephritis and septicaemia but no previous reports have documented meningitis secondary to this organism. Here we report what we believe to be the first clinical description of an adult with B. holmesii meningitis and bacteraemia with a brief review of published cases.

The spread of Mycoplasma pneumoniae is polyclonal in both an endemic setting in France and in an epidemic setting in Israel.

Mycoplasma pneumoniae infections occur both endemically and epidemically, and macrolide resistance has been spreading for 10 years worldwide. A substantial increased incidence of M. pneumoniae infections has been reported in several countries since 2010. Whether this increased incidence is attributed to different or to the same M. pneumoniae genotype is unknown. We have developed a multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) for the molecular typing of M. pneumoniae isolates. In this study, the MLVA typing method was modified and validated to be applicable directly to respiratory tract specimens without culture. This method was applied to 34 M. pneumoniae-positive specimens received at the Bordeaux Hospital, France, between 2007 and 2010 in an endemic setting, and to 63 M. pneumoniae-positive specimens collected during an epidemic surge of M. pneumoniae infections in 2010 in Jerusalem, Israel. The M. pneumoniae endemic spread was shown to be polyclonal in France, with 15 MLVA types identified. Strikingly, the Israeli epidemic surge was also a multi-clonal phenomenon, with 18 circulating MLVA types. The macrolide resistance-associated substitution, A2058G, was found in 22% of the Israeli patients. Macrolide-resistant M. pneumoniae belonged to four MLVA types, the MLVA type Z being the most frequent one. An association between the MLVA type Z and macrolide resistance might exist since macrolide resistance was present or generated during the course of illness in all patients infected with this MLVA type. In conclusion, the discriminatory power of the MLVA showed that the spread of M. pneumoniae strains in France in an endemic setting was polyclonal as well as the surge of M. pneumoniae infections in Israel in 2010.

Cluster of pseudoinfections with Burkholderia cepacia associated with a contaminated washer-disinfector in a bronchoscopy unit.

In December 2008, bronchoalveolar lavage fluid samples obtained from 3 patients were positive for Burkholderia cepacia complex on culture. Samples obtained from bronchoscopes and rinse-water samples obtained from the washer-disinfector were found to be positive for B. cepacia complex. The cause of this pseudo-outbreak was that the washer-disinfector was installed without the required antibacterial filter.

Characterization of biofilm formation by clinically relevant serotypes of group A streptococci.

Streptococcus pyogenes (group A streptococcus [GAS]) is a frequent cause of purulent infections in humans. As potentially important aspects of its pathogenicity, GAS was recently shown to aggregate, form intratissue microcolonies, and potentially participate in multispecies biofilms. In this study, we show that GAS in fact forms monospecies biofilms in vitro, and we analyze the basic parameters of S. pyogenes in vitro biofilm formation, using Streptococcus epidermidis as a biofilm-positive control. Of nine clinically important serotype strains, M2, M6, M14, and M18 were found to significantly adhere to coated and uncoated polystyrene surfaces. Fibronectin and collagen types I and IV best supported primary adherence of serotype M2 and M18 strains, respectively, whereas serotype M6 and M14 strains strongly bound to uncoated polystyrene surfaces. Absorption measurements of safranin staining, as well as electron scanning and confocal laser scanning microscopy, documented that primary adherence led to subsequent formation of three-dimensional biofilm structures consisting of up to 46 bacterial layers. Of note, GAS isolates belonging to the same serotype were found to be very heterogeneous in their biofilm-forming behavior. Biofilm formation was equally efficient under static and continuous flow conditions and consisted of the classical three steps, including partial disintegration after long-term incubation. Activity of the SilC signaling peptide as a component of a putative quorum-sensing system was found to influence the biofilm structure and density of serotype M14 and M18 strains. Based on the presented methods and results, standardized analyses of GAS biofilms and their impact on GAS pathogenicity are now feasible.

A streptococcal protease that degrades CXC chemokines and impairs bacterial clearance from infected tissues.

Group A Streptococcus (GAS) causes the life-threatening infection in humans known as necrotizing fasciitis (NF). Infected subcutaneous tissues from an NF patient and mice challenged with the same GAS strain possessed high bacterial loads but a striking paucity of infiltrating polymorphonuclear leukocytes (PMNs). Impaired PMN recruitment was attributed to degradation of the chemokine IL-8 by a GAS serine peptidase. Here, we use bioinformatics approach coupled with target mutagenesis to identify this peptidase as ScpC. We show that SilCR pheromone downregulates scpC transcription via the two-component system-SilA/B. In addition, we demonstrate that in vitro, ScpC degrades the CXC chemokines: IL-8 (human), KC, and MIP-2 (both murine). Furthermore, using a murine model of human NF, we demonstrate that ScpC, but not the C5a peptidase ScpA, is an essential virulence factor. An ScpC-deficient mutant is innocuous for untreated mice but lethal for PMN-depleted mice. ScpC degrades KC and MIP-2 locally in the infected skin tissues, inhibiting PMN recruitment. In conclusion, ScpC represents a novel GAS virulence factor functioning to directly inactivate a key element of the host innate immune response.

A locus of group A Streptococcus involved in invasive disease and DNA transfer.

Group A streptococcus (GAS) causes diseases ranging from benign to severe infections such as necrotizing fasciitis (NF). The reasons for the differences in severity of streptococcal infections are unexplained. We developed the polymorphic-tag-lengths-transposon-mutagenesis (PTTM) method to identify virulence genes in vivo. We applied PTTM on an emm14 strain isolated from a patient with NF and screened for mutants of decreased virulence, using a mouse model of human soft-tissue infection. A mutant that survived in the skin but was attenuated in its ability to reach the spleen and to cause a lethal infection was identified. The transposon was inserted into a small open reading frame (ORF) in a locus termed sil, streptococcal invasion locus. sil contains at least five genes (silA-E) and is highly homologous to the quorum-sensing competence regulons of Streptococcus pneumoniae. silA and silB encode a putative two-component system whereas silD and silE encode two putative ABC transporters. silC is a small ORF of unknown function preceded by a combox promoter. Insertion and deletion mutants of sil had a diminished lethality in the animal model. Virulence of a deletion mutant of silC was restored when injected together with the avirulent emm14-deletion mutant, but not when these mutants were injected into opposite flanks of a mouse. DNA transfer between these mutants occurred in vivo but could not account for the complementation of virulence. DNA exchange between the emm14-deletion mutant and mutants of sil occurred also in vitro, at a frequency of approximately 10-8 for a single antibiotic marker. Whereas silC and silD mutants exchanged markers with the emm14 mutant, silB mutant did not. Thus, we identified a novel locus, which controls GAS spreading into deeper tissues and could be involved in DNA transfer.