Cristina Belmar Campos

Prevalence and genotypes of extended spectrum beta-lactamases in Enterobacteriaceae isolated from human stool and chicken meat in Hamburg, Germany

Chicken meat has been proposed to constitute a source for extended spectrum beta-lactamase (ESBL)-carrying Enterobacteriaceae that colonize and infect humans. In this study the prevalence of ESBL-producing Enterobacteriaceae in stool samples from ambulatory patients who presented in the emergency department of the University Medical Centre Hamburg-Eppendorf with gastrointestinal complains and in chicken meat samples from the Hamburg region were analysed and compared with respect to ESBL-genotypes, sequence types and antibiotic resistance profiles. Twenty-nine (4.1%) of 707 stool samples and 72 (60%) of 120 chicken meat samples were positive for ESBL-producing Enterobacteriaceae. The distribution of ESBL genes in the stool vs. chicken meat isolates (given as % of total isolates from stool vs. chicken meat) was as follows: CTX-M-15 (38% vs. 0%), CTX-M-14 (17% vs. 6%), CTX-M-1 (17% vs. 69%), SHV-12 (3% vs. 18%) and TEM-52 (3% each). Comparison of ESBL- and multilocus sequence type revealed no correlation between isolates of human and chicken. Furthermore, ESBL-producing E. coli from stool samples were significantly more resistant to fluoroquinolones, aminoglycosides and/or trimethoprim-sulfamethoxazole than chicken isolates. The differences in ESBL-genotypes, sequence types and antibiotic resistance patterns indicate that in our clinical setting chicken meat is not a major contributor to human colonization with ESBL-carrying Enterobacteriaceae.

Yersinia Virulence Factor YopM Induces Sustained RSK Activation by Interfering with Dephosphorylation

Background Pathogenic yersiniae inject several effector proteins (Yops) into host cells, which subverts immune functions and enables the bacteria to survive within the host organism. YopM, whose deletion in enteropathogenic yersiniae results in a dramatic loss of virulence, has previously been shown to form a complex with and activate the multifunctional kinases PKN2 and RSK1 in transfected cells. Methodology/Principal Findings In a near physiological approach with double-affinity-tagged YopM being translocated into the macrophage cell line J774A.1 via the natural type three secretion system of Yersinia we verified the interaction of YopM with PKN2 and RSK1 and detected association with additional PKN and RSK isoforms. In transfected and infected cells YopM induced sustained phosphorylation of RSK at its activation sites serine-380 and serine-221 even in the absence of signalling from its upstream kinase ERK1/2, suggesting inhibition of dephosphorylation. ATP-depletion and in vitro assays using purified components directly confirmed that YopM shields RSK isoforms from phosphatase activity towards serines 380 and 221. Conclusions/Significance Our study suggests that during Yersinia infection YopM induces sustained activation of RSK by blocking dephosphorylation of its activatory phosphorylation sites. This may represent a novel mode of action of a bacterial virulence factor.

Identification of the Shiga Toxin-Producing Escherichia coli O104:H4 Strain Responsible for a Food Poisoning Outbreak in Germany by PCR

An outbreak caused by Escherichia coli serotype O104:H4 strains has been affecting northern Germany since May 2011, with 3,222 patients infected and 39 of them dead by 18 June ([4][1]). Around 25% of the patients developed hemolytic-uremic syndrome (HUS) ([1][2], [3][3]), a rate which is much higher

Duration of Fecal Shedding of Shiga Toxin–Producing Escherichia coli O104:H4 in Patients Infected During the 2011 Outbreak in Germany: A Multicenter Study

BACKGROUND In May-July 2011, Germany experienced a large food-borne outbreak of Shiga toxin 2-producing Escherichia coli (STEC O104:H4) with 3842 cases, including 855 cases with hemolytic uremic syndrome (HUS) and 53 deaths. METHODS A multicenter study was initiated in 5 university hospitals to determine pathogen shedding duration. Diagnostics comprised culture on selective media, toxin enzyme-linked immunosorbent assay, and polymerase chain reaction. Results were correlated with clinical and epidemiologic findings. Testing for pathogen excretion was continued after discharge of the patient. RESULTS A total of 321 patients (104 male, 217 female) were included (median age, 40 years [range, 1-89 days]). Median delay from onset of symptoms to hospitalization was 4 days (range, 0-17 days). Two hundred nine patients presented with HUS. The estimate for the median duration of shedding was 17-18 days. Some patients remained STEC O104:H4 positive until the end of the observation time (maximum observed shedding duration: 157 days). There was no significant influence of sex on shedding duration. Patients presenting with HUS had a significantly shortened shedding duration (median, 13-14 days) compared to non-HUS patients (median, 33-34 days). Antimicrobial treatment was also significantly associated with reduced shedding duration. Children (age≤15 years) had longer shedding durations than adults (median, 35-41 vs 14-15 days). CONCLUSIONS STEC O104:H4 is usually eliminated from the human gut after 1 month, but may sometimes be excreted for several months. Proper follow-up of infected patients is important to avoid further pathogen spread.

Extended spectrum beta-lactamase producing Enterobacteriaceae causing bloodstream infections in rural Ghana, 2007-2012.

BACKGROUND High prevalence of Extended Spectrum Beta-Lactamase (ESBL) producing Enterobacteriaceae threatens treatment options for invasive bloodstream infections in sub-Saharan Africa. OBJECTIVES To explore the frequency and genotype distribution of ESBL producing Enterobacteriaceae causing bloodstream infections in a primary health care setting in rural Ghana. METHODS Blood cultures from all patients with fever ≥38°C within 24h after admission (community-acquired) and from all neonates with suspected neonatal sepsis (hospital-acquired) were obtained. ESBL-producing isolates were characterized by combined disc test and by amplifying the blaCTX-M, blaTEM and blaSHV genes. Multilocus sequence typing (MLST) was performed for all ESBL-producing Klebsiella pneumoniae and Escherichia coli isolates, and all K. pneumoniae isolates were differentiated by pulsed-field gel electrophoresis (PFGE). RESULTS Among 426 Enterobacteriaceae isolated from blood cultures, non-typhoid Salmonella (n=215, 50.8%), S. Typhi (n=110, 26.0%), E. coli (n=50, 11.8%) and K. pneumoniae (n=41, 9.7%) were the most frequent. ESBL-producing isolates were restricted to the CTX-M-15 genotype and the species K. pneumoniae (n=34, 82.9%), Enterobacter cloacae complex (n=2, 66.7%) and E. coli (n=5, 10.0%). The rates of ESBL-producers in K. pneumoniae were 55.6% and 90.6% in community-acquired and neonatal bloodstream infections, respectively. MLST and PFGE analysis identified four outbreak clusters among neonates. CONCLUSIONS Considering the rural primary health care study setting, the high proportion of ESBL-producing Klebsiella pneumoniae is worrisome and might be devastating in the absence of second line antibiotics. Therefore, enhanced diagnostic laboratories for surveillance purposes and sustainable hospital hygiene measures must be considered to prevent further spread of multidrug resistant bacteria within rural communities.

Emergence of ceftazidime/avibactam non-susceptibility in an MDR Klebsiella pneumoniae isolate.

Background: Avibactam is a novel broad‐range &bgr;‐lactamase inhibitor active against Ambler class A (including ESBL and KPC) and some Ambler class C and D (e.g. OXA‐48) enzymes. We here report on the emergence of ceftazidime/avibactam resistance in clinical, multiresistant, OXA‐48 and CTX‐M‐14‐producing Klebsiella pneumoniae isolate DT12 during ceftazidime/avibactam treatment. Methods and results: Comparative whole‐genome sequence analysis identified two SNPs in the CTX‐M‐14‐encoding gene leading to two amino acid changes (P170S and T264I). Compared with WT CTX‐M‐14, expression of the CTX‐M‐14&Dgr;170&Dgr;264 isoform in Escherichia coli led to a >64‐ and 16‐fold increase in ceftazidime and ceftazidime/avibactam MICs, respectively, functionally linking the observed SNPs and elevated MICs. The mutated CTX‐M‐14 isoform exhibited augmented ceftazidime hydrolytic activity, which was a reasonable cause for impaired susceptibility to avibactam inhibition. The P170S exchange in CTX‐M‐14 was found in association with elevated ceftazidime/avibactam MICs for independent K. pneumoniae isolates, but was not sufficient for full resistance. Apparently, additional CTX‐M‐independent mechanisms contribute to ceftazidime/avibactam resistance in K. pneumoniae DT12. Conclusions: This study on the molecular basis of ceftazidime/avibactam resistance in clinical K. pneumoniae emerging in vivo underscores the need for continuous monitoring of ceftazidime/avibactam susceptibility during therapy. Despite sustained inhibition of OXA‐48, rapid development of CTX‐M‐14 isoforms exhibiting augmented ceftazidime hydrolytic activity may limit the usefulness of ceftazidime/avibactam monotherapies in infections caused by isolates carrying blaCTX‐M‐14 and blaOXA‐48.

Emergence of carbapenemases in Gram-negative bacteria in Hamburg, Germany

We analyzed a collection of carbapenem-resistant Gram-negative bacterial isolates and detected VIM-1, VIM-2, and KPC-2 in diverse enterobacterial species and Pseudomonas aeruginosa isolates. Our findings suggest a more widespread dissemination of carbapenemases in Germany than currently appreciated.

Genetic and biochemical characterization of HMB-1, a novel subclass B1 metallo-β-lactamase found in a Pseudomonas aeruginosa clinical isolate

Objectives To characterize a novel subclass B1 metallo-β-lactamase (MBL) found in an MDR Pseudomonas aeruginosa clinical isolate. Methods The isolate P. aeruginosa NRZ-03096 was recovered in 2012 from an anal swab from a patient hospitalized in Northern Germany and showed high MICs of carbapenems. MBL production was analysed by several phenotypic tests. Genetic characterization of the novel bla gene and MLST was performed by WGS. The novel bla gene was expressed in Escherichia coli TOP10 and the enzyme was subjected to biochemical characterization to determine the kinetic parameters K m and k cat . Results P. aeruginosa NRZ-03096 was resistant to all tested β-lactams and showed an MBL phenotype. Shotgun cloning experiments yielded a clone producing a novel subclass B1 enzyme with only 74.3% identity to the next nearest relative, KHM-1. The novel MBL was named HMB-1 (for Hamburg MBL). Analysis of WGS data showed that the bla HMB-1 gene was chromosomally located as part of a Tn 3 family transposon that was named Tn 6345 . Expression of bla HMB-1 in E. coli TOP10 led to increased resistance to β-lactams. Determination of K m and k cat revealed that HMB-1 had different hydrolytic characteristics compared with KHM-1, with lower hydrolytic rates for cephalosporins and a higher rate for imipenem. Conclusions The identification of HMB-1 further underlines the ongoing spread and diversification of carbapenemases in Gram-negative human pathogens and especially in P. aeruginosa .

Efficacy of introducing a checklist to reduce central venous line associated bloodstream infections in the ICU caring for adult patients

BackgroundCentral line-associated bloodstream infections (CLABSI) are a major source of sepsis in modern intensive care medicine. Some years ago bundle interventions have been introduced to reduce CLABSI. The use of checklists may be an additional tool to improve the effect of these bundles even in highly specialized institutions. In this study we investigate if the introduction of a checklist reduces the frequency of CLABSI.MethodsDuring the study period from October 2011 to September 2012, we investigated the effect of implementing a checklist for the placement of central venous lines (CVL). Patients were allocated either to the checklist group or to the control group, roughly in a 1:2 ratio. The frequency of CLABSI was compared between the two groups.ResultsDuring the study period 4416 CVL were inserted; 1518 in the checklist group and 2898 in the control group. The use of the checklist during CVL placement resulted in a lower CLABSI frequency. The incidence in the checklist group was 3.8 per 1000 catheter days as compared to 5.9 per 1000 catheter days in the control group (IRR = 0.57; p = 0.001). The use of the checklist also reduced the frequency of catheter colonisation significantly, 36.3 per 1000 catheter days in the checklist group vs 21.2 per 1000 catheter days in the control group, respectively (IRR = 0.58; p < 0.001).ConclusionThe introduction of a checklist to improve the adherence to hygiene standards while placement of central venous lines reduced the frequency of infections significantly.

Extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae in local and imported poultry meat in Ghana

Antibiotic use in animal husbandry has raised concerns on the spread of resistant bacteria. Currently animal products are traded globally with unprecedented ease, which has been challenging the control of antimicrobial resistance. This study aims to detect and characterize extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae from imported and locally produced poultry products sold in Ghana. Local and imported chicken meat was collected from 94 stores and markets throughout Kumasi (Ghana) and cultured on selective ESBL screening agar. Phenotypic ESBL-producing E. coli and K. pneumoniae isolates were confirmed by combined disc test and further characterized by antibiotic susceptibility testing, amplification of the blaCTX-M, blaTEM and blaSHV genes as well as multilocus sequence typing (MLST) and linked to the country of origin. Out of 200 meat samples, 71 (36%) samples revealed 81 ESBL-producing isolates (46 E. coli and 35 K. pneumoniae), with 44% (30/68) of local poultry and 31% (41/132) of imported products being contaminated. Most ESBL-producing isolates harboured the blaCTX-M-15 gene (61/81, 75%) and the dominant Sequence Types (ST) were ST2570 (7/35, 20%) among K. pneumoniae and ST10 (5/46, 11%) among E. coli. High numbers of ESBL-producing bacteria, particularly on local but also imported poultry meat, represent a potential source for human colonization and infection as well as spread within the community. Surveillance along the poultry production-food-consumer chain would be a valuable tool to identify sources of emerging multidrug resistant pathogens in Ghana.