Typing of Salmonella enterica serovar Infantis isolates from 51 outbreaks in Germany between 1974 and 2009 by a novel phage-typing scheme.

We developed a new phage-typing method and evaluated its application in combination with XbaI macrorestriction analysis by pulsed-field gel electrophoresis (PFGE) as a useful tool for the long-term epidemiology of Salmonella enterica serovar Infantis. In this study, we investigated 1008 S. Infantis isolates recovered from humans, various animal species and food products from 1973 to 2009. The typing scheme is based on 17 typing phages, defining 61 different patterns within the strain collection. The experiments showed that phage typing is a reliable method for differentiation of outbreaks and sporadic clinical cases as well as for elucidation of chains of transmission. The combined analysis of phage typing and PFGE revealed the existence of epidemic clones with a high stability over time like PT29/XB27 which was identified in nosocomial salmonellosis, community outbreaks as well as in broiler chickens from 2002 to 2009.

Antibiotic susceptibility profiles of neonatal invasive isolates of Escherichia coli from a 2-year nationwide surveillance study in Germany, 2009-2010.

A nationwide 2-year surveillance study on invasive neonatal Escherichia coli infections in Germany was conducted. A total of 158 isolates were tested for antibiotic susceptibility. The empirical treatment regimen of ampicillin plus gentamicin for neonatal sepsis appears to remain effective, but emerging resistance needs to be closely monitored.

High prevalence of extended-spectrum-ß-lactamase-producing Enterobacteriaceae in organic and conventional retail chicken meat, Germany.

OBJECTIVES: To determine the prevalence of extended-spectrum ß-lactamase (ESBL) production in Enterobacteriaceae in retail chicken meat in Germany. METHODS: A total of 399 chicken meat samples from nine supermarket chains, four organic food stores and one butcher's shop in two geographically distinct regions (Berlin and Greifswald) were screened for ESBL production using selective agar. Phenotypic ESBL isolates were tested for bla(TEM), bla(CTX-M) and bla(SHV) genes using PCR and DNA sequencing. Antibiotic coresistances were determined and strain typing was performed using PCR-based phylogenetic grouping and XbaI-PFGE. RESULTS: A total of 185 confirmed ESBL isolates were obtained from 175 samples (43.9%) from all tested sources. The majority of isolates were Escherichia coli producing ESBL types SHV-12 (n = 82), CTX-M-1 (n = 77) and TEM-52 (n = 16). No differences could be observed in the prevalence of ESBL producers between organic and conventional samples. 73.0% of the ESBL producers showed coresistance to tetracycline, 35.7% to co-trimoxazole and 7.6% to ciprofloxacin. Strain typing of selected E. coli isolates from Berlin revealed identical macrorestriction patterns for several isolates from samples taken from the same stores. CONCLUSIONS: This is the first comprehensive study from Germany showing a high prevalence of TEM-, CTX-M- and SHV-type ESBLs in Enterobacteriaceae isolated from retail chicken meat. The high rate of coresistance to different classes of antibiotics in the ESBL producers might reflect the common veterinary usage of these and related substances. There is an urgent need to further evaluate the role of poultry in the transmission of highly resistant ESBL-producing bacteria in humans.

[Current data and trends about the resistance of Gram-negative pathogens to beta-lactams]. / Aktuelle Daten und Trends zur ß-Lactam-Resistenz bei gramnegativen Infektionserregern.

In recent years the resistance of Gram-negative pathogens to beta-lactam antibiotics, such as cephalosporins and carbapenems has increased. The resistant strains produce different beta-lactam hydrolysing enzymes (beta-lactamases). In particular extended spectrum beta-lactamases (ESBL) are prevalent in Escherichia coli and Klebsiella pneumoniae. The ESBL genes are located on different plasmids facilitating the transfer of resistance within a species and between different Gram-negative species. Within the scope of various studies the Robert Koch Institute in Wernigerode investigated ESBL-producing human Enterobacteriaceae using molecular methods. The results showed that distinct ESBL types, such as the CTX-M enzymes are dominant in Germany whereby CTX-M-15 and CTX-M-1 are the most prevalent variants in E. coli and K. pneumoniae. The aim of ongoing investigations within the RESET network project is to investigate the dissemination pathways of ESBL-producing bacteria in different settings (e.g. in humans, animals and food).

Evaluation of the VITEK 2 AST-N111 card for detection of extended-spectrum beta-lactamases (ESBLs) in Escherichia coli, Klebsiella pneumoniae, and Klebsiella oxytoca compared to ESBL Etests and combination disk methods.

The VITEK 2 AST-N111 card was evaluated for detection of extended-spectrum beta-lactamases (ESBLs) by testing 51 ESBL positive and 50 ESBL negative isolates of E. coli, K. pneumoniae, and K. oxytoca. The occurrence of beta-lactamase genes was confirmed by PCR and sequencing. The advanced expert system (AES) of the VITEK 2 system achieved sensitivity and specificity values of 100% and 96.0%, respectively. The ESBL test of the VITEK 2 AST-N111 card showed a sensitivity of 92.1% and a specificity of 90.0%. Contradictory results obtained with the two VITEK 2 tools could be clarified by combination disk tests in nine of 11 isolates. The combined use of AES and ESBL tests of the AST-N111 card in association with combination disk tests in case of contradictory results seems to be a reliable method for ESBL detection.

Carbapenem-non-susceptible Enterobacteriaceae in Europe: conclusions from a meeting of national experts.

The emergence and global spread of carbapenemase-producing Enterobacteriaceae is of great concern to health services worldwide. These bacteria are often resistant to all beta-lactam antibiotics and frequently co-resistant to most other antibiotics, leaving very few treatment options. The epidemiology is compounded by the diversity of carbapenem-hydrolysing enzymes and the ability of their genes to spread between different bacterial species. Difficulties are also encountered by laboratories when trying to detect carbapenemase production during routine diagnostic procedures due to an often heterogeneous expression of resistance. Some of the resistance genes are associated with successful clonal lineages which have a selective advantage in those hospitals where antimicrobial use is high and opportunities for transmission exist; others are more often associated with transmissible plasmids. A genetically distinct strain of Klebsiella pneumoniae sequence type (ST) 258 harbouring the K. pneumoniae carbapenemases (KPC) has been causing epidemics of national and international proportions. It follows the pathways of patient referrals, causing hospital outbreaks along the way. Simultaneously, diverse strains harbouring New Delhi metallo-beta-lactamase (NDM-1) are repeatedly being imported into Europe, commonly via patients with prior medical exposure in the Indian subcontinent. Since the nature and scale of carbapenem-non-susceptible Entrobacteriaceae as a threat to hospital patients in Europe remains unclear, a consultation of experts from 31 countries set out to identify the gaps in diagnostic and response capacity, to index the magnitude of carbapenem-non-susceptibility across Europe using a novel five-level staging system, and to provide elements of a strategy to combat this public health issue in a concerted manner

Infections caused by extended-spectrum ß-lactamase-producing Enterobacterales after rectal colonization with ESBL-producing Escherichia coli or Klebsiella pneumoniae.

OBJECTIVES: Infections as a result of extended-spectrum ß-lactamase-producing Enterobacterales (ESBL-E) are considered infections with a high public health burden. In this study, we aimed to identify incidences of and risk factors for healthcare-associated infections (HAIs) after rectal colonization with ESBL-producing Escherichia coli (ESBL-EC) or Klebsiella pneumoniae (ESBL-KP). METHODS: This prospective cohort study was performed in 2014 and 2015. Patients colonized with ESBL-EC or ESBL-KP were monitored for subsequent HAI with ESBL-E and other pathogens. In the case of an ESBL-E infection, rectal and clinical isolates were compared using pulsed-field gel electrophoresis (PFGE), and whole-genome sequencing (WGS) for ESBL-KP isolates. Proportional hazard models were applied to identify risk factors for HAIs, and to analyse competing risks. RESULTS: Among all patients admitted to the hospital during the study period, 13.6% were rectally screened for third-generation cephalosporin-resistant Enterobacterales (3GCREB). A total of 2386 rectal carriers of ESBL-EC and 585 of ESBL-KP were included in the study. Incidence density (ID) for HAI with ESBL-E was 2.74 per 1000 patient days at risk (95% confidence interval (CI) 2.16-3.43) among carriers of ESBL-EC, while it was 4.44 per 1000 patient days at risk (95% CI 3.17-6.04) among carriers of ESBL-KP. In contrast, ID for HAI with other pathogens was 4.36 per 1000 patient days at risk (95% CI 3.62-5.21) among carriers of ESBL-EC, and 5.00 per 1000 patient days at risk (95% CI 3.64-6.69) among carriers of ESBL-KP. Cox proportional hazard regression analyses identified colonization with ESBL-KP (HR = 1.58, 95% CI 1.068-2.325) compared with ESBL-EC as independent risk factor for HAI with ESBL-E. The results were consistent over all competing risk analyses. CONCLUSIONS: Clinicians should be aware of the increased risk of ESBL-E infections among patients colonized with ESBL-KP compared with ESBL-EC that might be caused by underlying diseases, higher pathogenicity of ESBL-KP and other factors.

Evaluation of six commercial products for colistin susceptibility testing in Enterobacterales.

OBJECTIVES: The recommended technique for colistin susceptibility testing by both EUCAST and CLSI is broth microdilution (BMD). However, many routine laboratories still use other methods such as gradient strips or semi-automated systems. The objective of this study was to compare six of the most widespread commercial products for colistin susceptibility testing in Europe with in-house prepared BMD. METHODS: A collection of 325 carbapenemase-producing Enterobacterales was tested for colistin susceptibility with three semi-automated systems (Vitek 2, BD Phoenix, MicroScan WalkAway), one gradient-strip test (Etest®) and two commercial BMD products (MICRONAUT-S, TREK Sensititre). BMD, in-house prepared according to ISO standard 20776-1, served as reference. RESULTS: The MICRONAUT-S BMD performed best with only one false-resistant (major error, ME) and four false-susceptible (very major error, VME) results while the TREK BMD performed poorer with 16 ME and seven VME. The semi-automated systems Vitek 2 and Phoenix performed poorly with 31 and 26 VME, respectively. The WalkAway semi-automated system showed 16 and 13 false results, depending on the inoculation method. The Etest® showed six ME and 10 VME. CONCLUSIONS: This study shows that colistin susceptibility testing remains a challenging task for laboratories. It emphasizes the need for selecting the most reliable test method to advocate proper treatment and shows that critical evaluation and precautious usage of colistin susceptibility testing results is constantly required.

Prevalence and risk factors for colonization by Clostridium difficile and extended-spectrum ß-lactamase-producing Enterobacteriaceae in rehabilitation clinics in Germany.

BACKGROUND: Rehabilitation clinics may vary widely in terms of type of care provided, duration of hospital stay, and case severity. Few data are available on prevalence of Clostridium difficile or extended-spectrum ß-lactamase-producing Enterobacteriaceae (ESBL-E) colonization in rehabilitation clinics in Germany. AIM: This study investigated the frequency of intestinal colonization by these pathogens among patients in rehabilitation clinics of different specialization. METHODS: In the scope of a point prevalence study, faecal samples and demographic and clinical data were collected in five rehabilitation clinics. Samples were screened for C. difficile and ESBL-E by culture. Isolates were characterized by polymerase chain reaction for C. difficile toxins A and B, for ß-lactamase genes, and by molecular typing including pulsed-field gel electrophoresis and PCR-based ribotyping. FINDINGS: Of 305 patients screened, 11.1% were colonized by toxigenic C. difficile and 7.5% by ESBL-E. Colonization rates differed markedly between facilities, ranging from 1.6% to 26.3% for C. difficile and from zero to 23.7% for ESBL-E. Prevalence of colonization by C. difficile and ESBL-E was higher in neurological rehabilitation clinics than in clinics with other specialties (P<0.001). Molecular typing revealed six patients from one neurological rehabilitation clinic harbouring a unique C. difficile strain (ribotype 017). CTX-M-15 was the most prevalent ESBL type. We detected several indistinguishable pairs of ESBL-E isolates within some facilities. CONCLUSION: Significant differences were found in the prevalence of C. difficile and ESBL-E between rehabilitation clinics. Facilities providing specialized medical care for critically ill patients had higher prevalence rates. These results may help to delineate the requirements for infection prevention and control in rehabilitation clinics.

Recurrent outbreaks caused by the same Salmonella enterica serovar Infantis clone in a German rehabilitation oncology clinic from 2002 to 2009.

BACKGROUND: Repeated outbreaks of salmonellosis caused by Salmonella enterica serovar Infantis at a rehabilitation clinic in Germany were investigated microbiologically from August 2002 to August 2009. AIM: To identify the sources of transmission and characterize the S. enterica serovar Infantis isolates. METHODS: Associated with these outbreaks, isolates from 98 patients, two kitchen staff, five food samples, four swabs of kitchen facilities, three samples of chicken faeces and one sample of sewage water were evaluated by phage typing. All S. enterica serovar Infantis isolates investigated (N=113) were related to phage type (PT) 29. Additionally, 44 of the 113 isolates were selected at random for typing by XbaI macrorestriction and pulsed-field gel electrophoresis (PFGE). FINDINGS: Typing of the 44 isolates showed that the recurrent infections were caused by the single clone PT 29/XB27+44 (42/44, 95.5%). The most likely route of transmission was only identified in the last outbreak in 2009 within the present study. It was found to be cross-contamination in the kitchen facilities (emanating from a contaminated wooden panel), in combination with carriers among the kitchen staff. CONCLUSIONS: This study demonstrated important details of hospital-specific epidemiological processes, and alludes to a long-term reservoir of an epidemic clone of S. enterica serovar Infantis either in a backyard flock of poultry or in an inanimate kitchen reservoir.

Serotonin and 5-hydroxyindoleacetic acid in fertile and subfertile men.

High levels of serotonin and 5-HIAA were found in the urine of a group of 102 oligospermic and azoospermic men. These levels were significantly higher than those of normal fertile men.

The use of antiserotonin-cyproheptadine HCL in pregnancy: an experimental and clinical study.

PIP: Habitual abortion of psychogenic origin may be associated with increased serotonin production. Serotonin is metabolized by monoamin oxidase (MAO) to hydroxyindole acetic acid (5-HIAA) which is excreted in the urine. When this metabolic system is inadequate excess serotonin may cause contractions of the estrogen-sensitized myometrium and, thus, abortion. In normal pregnancy and in nonpregnant women urinary 5-HIAA is 5.6 mg/day. Women suffering from habitual abortion os psychogenic origin showed 5-HIAA excretion of 10.6 mg/day during pregnancy with high amounts of nonmetabolized serotonin. In rats injection of pargyline HC1, a MAO inhibitor, produced abortion in 84%; a serotonin antagonist, cyproheptadine HC1, injected sc 4-8 hours prior to MAO inhibitor treatment preserved 42% of fetuses. No toxicity was shown in 92.8% of rat fetuses in a teratogenic study. Later 29 women with a total of 31 pregnancies were given 4 to 16 mg/day cyproheptadine HC1 for several months starting early in pregnancy. Previous study had revealed no other causes to explain their spontaneous abortions and treatments with progesterone, antibiotics, and vitamins in earlier pregnancies had been unsuccessful. After cyproheptadine HC1 treatment there were 3 spontaneous abortions, 23 normal infants delivered at term, and 5 premature deliveries of which 1 was a stillbirth. No teratogenic effects were observed. Reports by others in similar cases and results in a control group of 19 women in this study all give much higher rates of spontaneous abortion. Side effects were few and yielded to reduced dosage. It is recommended that patients suffering habitual abortion be examined for serotonin and 5-HIAA excretion. When indicated, treatment with antiserotonin drugs should be given. Observations indicate serotonin production or metabolism may be the decisive factor relating emotional stress to abortion.^ieng

Effect of manual lymphdrainage massage on blood components and urinary neurohormones in chronic lymphedema.

In an earlier paper we have shown that manual lymph drainage massage of edematous limbs can result in the excretion of up to 1 liter urine derived from reabsorption and transport from the interstitial fluid, simultaneously with significant changes in the excretion of urinary neurohormones. These findings indicated that histamine and serotonin were released from the edematous tissue and that circulation improved through increased output of adrenaline and noradrenaline. The results achieved led us to assume that similar changes may have occurred in the blood during treatment, and induced us to study the effect of manual lymphdrainage on various blood constituents and urinary neurohormones.

Effect of manual lymph drainage massage on urinary excretion of neurohormones and minerals in chronic lymphedema.

Treatment of 29 cases of chronic lymphedema of various origins, mostly of the lower limbs, by manual lymph drainage massage resulted in significant changes of neurohormone excretion in the urine, whereas the secretion of 17-KS, thyroxine, minerals, and creatinine was not significantly changed. Comparison of the values of urinalysis before and after manual lymph drainage of the patients showed the following changes: 17-KS; -3.5% (non significant); 17-OH: -31% (significant); adrenaline: +50% (significant); noradrenaline: +19% (significant); serotonin: -22% (significant); 5-HIAA: +21% (significant); histamine: +129% (highly significant); thyroxine: -17% (nonsignificant); creatinine: -17% (nonsignificant); sodium: -1% (nonsignificant); potassium: -14% (nonsignificant). The corresponding values for ten controls were all non significant. These findings underline the importance of adrenaline and noradrenaline release by manual lymph drainage, which improves circulation. On the other hand, our results indicate the involvement of histamine and perhaps of serotonin in lymphedema formation, and suggest a combination of manual lymph drainage massage with antihistamine and antiserotonin treatment.

Serotonin in the portal vein after acidification.

To test whether serotonin release from the argentaffin cells of the intestinal tract can elicit peptic ulcer, rats were given intraduodenal infusions of N/10 and N/20 hydrochloric acid. Serotonin measured in the portal venous blood of rats which had had an intraduodenal infusion of N/20 HCl for five minutes was significantly higher than in the portal (P less than 0.05) or systemic (P less than 0.01) blood of control animals. After longer intervals serotonin release tapered off and measurements obtained 30 minutes after an infusion of N/10 HCl did not differ significantly from those in the controls.

A novel IS26 structure surrounds blaCTX-M genes in different plasmids from German clinical Escherichia coli isolates.

This report focuses on the molecular characterization of 22 extended-spectrum beta-lactamase-producing Escherichia coli isolates collected in a German university hospital during a period of 9 months in 2006. Relationship analysis of clinical isolates was done via PFGE, multilocus sequence typing, plasmid profiling and additionally PCR for bla(ESBL) detection and determination of phylogroups. After conjugal transfer, plasmid isolation and subsequent PCR for bla(ESBL) detection and determination of incompatibility groups were performed. Using one-primer walking, up to 3600 bp upstream and downstream of different bla(CTX-M) genes could be sequenced. beta-Lactamases found were TEM-1 (n=14), SHV-5 (n=1) and a wide variety of CTX-M types (n=21), i.e. CTX-M-15 (n=12), CTX-M-1 (n=4), CTX-M-14 (n=2), CTX-M-9 (n=1), CTX-M-3 (n=1) and one new type, CTX-M-65 (n=1). In 18 isolates, bla(ESBL) genes were located on conjugative plasmids of sizes between 40 and 180 kbp belonging to incompatibility groups FII (n=9), N (n=5) and I1 (n=4). bla(CTX-M) was found to be associated with the common elements ISEcp1, IS26 and IS903-D, but with unusual spacer sequences for ISEcp1 in two isolates. These insertion sequences, connected to bla(CTX-M) as well as other genes, were located between two IS26 elements in a configuration that has not yet been described. The results reveal the emergence of bla(ESBL), predominantly bla(CTX-M), located on different plasmids harboured by genotypically different E. coli strains. The identical gene arrangement in the bla(CTX-M) neighbourhood in plasmids of different incompatibility groups indicates a main role of IS26 in distribution of mobile resistance elements between different plasmids.

Emergence of CTX-M-15 extended-spectrum beta-lactamase-producing Klebsiella pneumoniae isolates in Bosnia and Herzegovina.

Fifty-seven nosocomial Klebsiella pneumoniae isolates producing extended-spectrum beta-lactamases (ESBLs) were collected between February 2007 and November 2007 in different wards of the Sarajevo (Bosnia-Herzegovina) reference hospital. These isolates comprise two major epidemic pulsed-field electrophoresis-defined clones plus two minor clones. In addition to the ESBL-mediated resistance, all strains uniformly showed resistance to ciprofloxacin, gentamicin and tobramycin. The beta-lactamases involved in this resistance phenotype were TEM-1, SHV-1, and CTX-M-15, as demonstrated by isoelectric focusing, PCR amplification, and sequencing. TEM-1 and CTX-M-15 beta-lactamases, as well as the aminoglycoside resistance determinants, were encoded in plasmids that could be transferred to Escherichia coli by conjugation. In three of the infected patients with the predominant clone, cefoxitin resistance development (MICs >128 mg/L) was documented. The analysis of the outer membrane proteins of the cefoxitin-susceptible and cefoxitin-resistant isolates revealed that the former expressed only one of the two major porins, OmpK36, whereas in the latter, the expression of Ompk36 was altered or abolished. This is the first report of CTX-M-15-producing K. pneumoniae in Bosnia-Herzegovina. Furthermore, we document and characterize for the first time cefoxitin resistance development in CTX-M-15-producing K. pneumoniae.