Amir Zlotkin

Potential role of active surveillance in the control of a hospital-wide outbreak of carbapenem-resistant Klebsiella pneumoniae infection.

BACKGROUND The recent emergence of carbapenem resistance among Enterobacteriaceae is a major threat for hospitalized patients, and effective strategies are needed. OBJECTIVE To assess the effect of an intensified intervention, which included active surveillance, on the incidence of infection with carbapenem-resistant Klebsiella pneumoniae. SETTING Sheba Medical Center, a 1,600-bed tertiary care teaching hospital in Tel Hashomer, Israel. DESIGN Quasi-experimental study. METHODS The medical records of all the patients who acquired a carbapenem-resistant K. pneumoniae infection during 2006 were reviewed. An intensified intervention was initiated in May 2007. In addition to contact precautions, active surveillance was initiated in high-risk units. The incidence of clinical carbapenem-resistant K. pneumoniae infection over time was measured, and interrupted time-series analysis was performed. RESULTS The incidence of clinical carbapenem-resistant K. pneumoniae infection increased 6.42-fold from the first quarter of 2006 up to the initiation of the intervention. In 2006, of the 120 patients whose clinical microbiologic culture results were positive for carbapenem-resistant K. pneumoniae, 67 (56%) developed a nosocomial infection. During the intervention period, the rate of carbapenem-resistant K. pneumoniae rectal colonization was 9%. Of the 390 patients with carbapenem-resistant K. pneumoniae colonization or infection, 204 (52%) were identified by screening cultures. There were a total of 12,391 days of contact precautions, and of these, 4,713 (38%) were added as a result of active surveillance. After initiation of infection control measures, we observed a significant decrease in the incidence of carbapenem-resistant K. pneumoniae infection. CONCLUSIONS The use of active surveillance and contact precautions, as part of a multifactorial intervention, may be an effective strategy to decrease rates of nosocomial transmission of carbapenem-resistant K. pneumoniae colonization or infection.

Cloning of the koi herpesvirus (KHV) gene encoding thymidine kinase and its use for a highly sensitive PCR based diagnosis.

BackgroundOutbreaks with mass mortality among common carp Cyprinus carpio carpio and koi Cyprinus carpio koi have occurred worldwide since 1998. The herpes-like virus isolated from diseased fish is different from Herpesvirus cyprini and channel catfish virus and was accordingly designated koi herpesvirus (KHV). Diagnosis of KHV infection based on viral isolation and current PCR assays has a limited sensitivity and therefore new tools for the diagnosis of KHV infections are necessary.ResultsA robust and sensitive PCR assay based on a defined gene sequence of KHV was developed to improve the diagnosis of KHV infection. From a KHV genomic library, a hypothetical thymidine kinase gene (TK) was identified, subcloned and expressed as a recombinant protein. Preliminary characterization of the recombinant TK showed that it has a kinase activity using dTTP but not dCTP as a substrate. A PCR assay based on primers selected from the defined DNA sequence of the TK gene was developed and resulted in a 409 bp amplified fragment. The TK based PCR assay did not amplify the DNAs of other fish herpesviruses such as Herpesvirus cyprini (CHV) and the channel catfish virus (CCV). The TK based PCR assay was specific for the detection of KHV and was able to detect as little as 10 fentograms of KHV DNA corresponding to 30 virions. The TK based PCR was compared to previously described PCR assays and to viral culture in diseased fish and was shown to be the most sensitive method of diagnosis of KHV infection.ConclusionThe TK based PCR assay developed in this work was shown to be specific for the detection of KHV. The TK based PCR assay was more sensitive for the detection of KHV than previously described PCR assays; it was as sensitive as virus isolation which is the golden standard method for KHV diagnosis and was able to detect as little as 10 fentograms of KHV DNA corresponding to 30 virions.

Recovery of Streptococcus iniae from diseased fish previously vaccinated with a streptococcus vaccine

ABSTRACT Streptococcus iniae was recovered from diseased rainbow trout (Oncorhynchus mykiss, Walbaum) previously vaccinated against streptococcosis. PCR and serological methods indicate the presence of a new serotype in the diseased fish.

Trojan horse effect: phagocyte-mediated Streptococcus iniae infection of fish.

ABSTRACT The salmonid macrophage-like cell line RTS-11 and purified trout pronephros phagocytes were used to analyze in vitro entry and survival of two Streptococcus iniae serotypes. Efficient invasion by S. iniae occurred in both cells, but only the type II strain persisted in pronephros phagocytes for at least 48 h. Ex vivo models of opsonin-dependent phagocytosis by pronephros phagocytes demonstrated increased phagocytosis efficacy. Analysis of phagocytes collected from diseased fish demonstrated that ∼70% of the bacteria contained in the blood during the septic phase of the disease were located within phagocytes, suggesting an in vivo intracellular lifestyle. In addition to the augmented levels of bacteremia and enhanced survival within phagocytes, S. iniae type II induces considerable apoptosis of phagocytes. These variabilities in intramacrophage lifestyle might explain differences in the outcomes of infections caused by different serotypes. The generalized septic disease associated with serotype II strains is linked not only to the ability to enter and multiply within macrophages but also to the ability to cause considerable death of macrophages via apoptotic processes, leading to a highly virulent infection. We assume that the phenomenon of survival within phagocytes coupled to their apoptosis plays a crucial role in S. iniae infection. In addition, it may provide the pathogen an efficient mechanism of translocation into the central nervous system.

Clonality and Diversity of the Fish Pathogen Lactococcus garvieae in Mediterranean Countries

ABSTRACT Infection with Lactococcus garvieae is considered the most important risk factor for the European trout industry, and the losses are approximately 50% of the total production. To improve our understanding of the genetic links among strains originating from different countries, we examined the population structure of L. garvieae by comparing 81 strains isolated from different sources and ecosystems (41 farms in six countries) in which the bacterium is commonly found. Genetic similarities (as assessed with molecular tools, including restriction fragment length polymorphism ribotyping with two endonucleases) were compared with serological data. The combined results reveal that in endemic sites the bacterial population displays a clonal structure, whereas bacterial diversity characterizes sites where the infection is sporadic.

Emergence of Novel Streptococcus iniae Exopolysaccharide-Producing Strains following Vaccination with Nonproducing Strains

ABSTRACT Streptococcus iniae is a major pathogen of fish, producing fatal disease among fish species living in very diverse environments. Recently, reoccurrences of disease outbreaks were recorded in rainbow trout (Oncorhynchus mykiss, Walbaum) farms where the entire fish population was routinely vaccinated. New strains are distinguished from previous strains by their ability to produce large amounts of extracellular polysaccharide that is released into the medium. Present findings indicate that the extracellular polysaccharide is a major antigenic factor, suggesting an evolutionary selection of strains capable of extracellular polysaccharide production.

An outbreak of Burkholderia cenocepacia bacteremia in immunocompromised oncology patients

BackgroundBurkholderia cepacia is a common environmental bacterium that is resistant to disinfectants, and therefore is often encountered as a hospital-acquired pathogen. We describe an outbreak of B. cenocepacia bacteremia among hospitalized oncology patients.MethodsA matched case–control study and an extensive environmental investigation were conducted. Species were identified by RFLP of the amplified recA gene. DNA was fingerprinted by pulsed-field gel electrophoresis (PFGE).ResultsBetween November 2005 and September 2006, B. cenocepacia bacteremia developed in 17 patients with underlying malignancy of whom 14 had tunneled central venous catheters. All patients had fever and chills which subsided following removal of the central catheter and administration of ceftazidime. Extensive epidemiological investigation could not find a common source for the outbreak. Patients were hospitalized in three different buildings with different health care personnel. Medications were prepared in different sites by different personnel. A multivariate analysis demonstrated that the independent risk factors for developing nosocomial B. cenocepacia bacteremia were hospitalization at the center for long-term support (OR 28.8; 95% CI 1.83–453.4) and reduced use of antibiotics during the last month (OR 0.07; 95% CI 0.01–0.40). All isolates had identical antimicrobial susceptibility; PFGE indicated that a complex of closely related strains was involved in the outbreak. All isolates were identified as B. cenocepacia, known to infect cystic fibrosis patients. Strict infection control measures terminated the outbreak.ConclusionsB. cenocepacia is an emerging nosocomial pathogen among oncology patients.

A pivotal role for the Streptococcus iniae extracellular polysaccharide in triggering proinflammatory cytokines transcription and inducing death in rainbow trout

Streptococcus iniae is a major pathogen of fish, causing considerable economic losses in Israel, the United States and the Far East. Containment of mortalities through vaccination was recently compromised due to the emergence of novel vaccine-escape strains that are distinguished from previous strains by their ability to produce large amounts of extracellular polysaccharide (EPS) that is released to the medium. In vitro and in vivo data now indicate that the EPS is a major virulence factor, capable of triggering the proinflammatory cytokine machinery and inducing mortality of fish. Streptococcus iniae EPS might therefore be considered to be responsible for sepsis and death just as lipopolysaccharide is for Gram-negative pathogens.

Helicobacter pylori DNA in dental plaques, gastroscopy, and dental devices.

The role of dental plaque in the transmission of Helicobacter pylori (Hp) is unclear due to variability in the detection rates and techniques used. We used nested PCR to estimate the incidence of Hp in dental plaques of 24 dental hygienists. We found an unexpectedly high incidence (50%) of Hp DNA in dental plaques using sterilized dental probes. Additional treatment of sonication and SDS wash prior to sterilization of dental probes reduced the incidence to 13%. We used the treated probes to assess Hp presence in plaque samples of 47 patients visiting the dental clinic for teeth cleaning. Hp DNA was detected in 24% of cases. Since these data may reflect instrument contamination, we tested dental probes, endoscopes, and endoscopy forceps and found that 12.5–37.5% of them were contaminated. Consequently, dental plaques may be a candidate reservoir for Hp, medical equipment may contribute to Hp transmission, and sample collection techniques can bias the true prevalence of Hp in a population.

Molecular features of heterogeneous vancomycin-intermediate Staphylococcus aureus strains isolated from bacteremic patients

BackgroundHeterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) bacteremia is an emerging infection. Our objective was to determine the molecular features of hVISA strains isolated from bacteremic patients and to compare them to methicillin resistant S. aureus (MRSA) and methicillin sensitive S. aureus (MSSA) blood isolates.ResultsWe assessed phenotypic and genomic changes of hVISA (n = 24), MRSA (n = 16) and MSSA (n = 17) isolates by PCR to determine staphylococcal chromosomal cassette (SCCmec) types, Panton-Valentine leukocidin (PVL) and the accessory gene regulator (agr) loci. Biofilm formation was quantified. Genetic relatedness was assessed by PFGE. PFGE analysis of isolates was diverse suggesting multiple sources of infection. 50% of hVISA isolates carried SCCmec type I, 21% type II; 25% type V; in 4% the SCCmec type could not be identified. Among MRSA isolates, 44% were SCCmec type I, 12.5% type II, 25% type V, 12.5% were non-typable, and 6% were SCCmec type IVd. Only one hVISA isolate and two MSSA isolates carried the PVL. Biofilm formation and agr patterns were diverse.ConclusionhVISA isolates were diverse in all parameters tested. A considerable number of hVISA and MRSA strains carried the SCCmec type V cassette, which was not related to community acquisition.