Emergence and spread of moxifloxacin-resistant Clostridium difficile ribotype 231 in Sweden between 2006 and 2015.

An aggregation of moxifloxacin-resistant Clostridium difficile ribotype 231 (RT231) isolates was first identified in the county of Stockholm in 2008, and by the end of 2015 isolates of RT231 had spread to 13 of 21 Swedish counties. We investigated the epidemiology of C. difficile RT231 in Sweden between 2006 and 2015 using whole genome sequencing (WGS) and evaluated whether its emergence could be associated with extended moxifloxacin use. We performed WGS and phylogenetic analysis of 51 C. difficile RT231 strains isolated in Sweden over a 10-year period. We also calculated the county-specific prescription rates for moxifloxacin between 2005 and 2015. Using WGS and detailed single nucleotide polymorphism analysis, we demonstrated three divergent sublineages of moxifloxacin-resistant C. difficile RT231 in Sweden from 2008 to 2015. A set of closely related RT231 was identified in hospitals located in the counties of Stockholm and Uppsala in 2008. Another set of RT231 isolates was found in four different counties in the Uppsala-Örebro Health Care Region. A gradual drop in moxifloxacin use in the county of Stockholm coincided with a reduction of RT231 in the area. However, RT231 continued to be frequent in surrounding counties including Uppsala, a county that also had the highest moxifloxacin prescription rates. We demonstrated frequent transmission of C. difficile RT231 within and between counties, indicating the importance of careful monitoring of hospitalized individuals infected with moxifloxacin-resistant C. difficile as well as the need for a strict moxifloxacin prescription policy.

IgG antibody response to toxins A and B in patients with Clostridium difficile infection.

IgG antibodies against Clostridium difficile toxins A and B were followed in controls and in patients with an initial C. difficile infection (CDI). Of the 50 CDI patients, 38 were cured and 12 developed recurrence. Compared to controls, patients had significantly lower anti-toxin A and B IgGs at inclusion, but the subsequent levels rose slightly regardless of clinical outcome. The results imply that the general serum reactivity against toxins A and B in the population reduces the risk of CDI, which suggests implications for vaccine strategies.

Geographical clustering of cases of infection with moxifloxacin-resistant Clostridium difficile PCR-ribotypes 012, 017 and 046 in Sweden, 2008 and 2009.

We report the results of two nationwide surveillance studies of Clostridium difficile infection conducted during 2008 and 2009 in Sweden. The first study aimed to identify and quantify the proportion of C. difficile isolates with decreased susceptibility to moxifloxacin, particularly those of PCR-ribotype 027. From December 2007 to September 2008, 20 of 28 regional laboratories sent 585 isolates to the Swedish Institute for Infectious Disease Control for typing. A majority of the isolates (454 of 585; 78%) belonged to four PCR ribotypes (012, SE37, 017 and 046), all clustered in geographical regions. Only two type 027 isolates were found, both from the same patient. In the second study, involving all 28 regional laboratories, all consecutive C. difficile isolates collected during two time periods in 2009 (n=364) were typed and tested for susceptibility to clindamycin, erythromycin, moxifloxacin, metronidazole and vancomycin. The three most common PCR ribotypes were SE21, 001 and 020 (22% of all isolates). Types 012, 017, and 046 were geographically clustered and associated with decreased susceptibility to moxifloxacin, clindamycin and erythromcin. The extent of moxifloxacin prescription was highly variable among counties, indicating a need for careful monitoring of prescription rates to follow its role in C. difficile epidemiology.

In vitro susceptibility to 17 antimicrobials of clinical Clostridium difficile isolates collected in 1993-2007 in Sweden.

This study investigated the MICs of 17 antimicrobials, for 606 toxigenic clinical isolates of Clostridium difficile collected between 1993 and 2007 in Sweden. Low MIC(90) values were found for metronidazole (0.5 mg/L), vancomycin (1.0 mg/L), teicoplanin (0.125 mg/L), fusidic acid (1.0 mg/L), linezolid (2.0 mg/L), daptomycin (2.0 mg/L) and tigecycline (0.064 mg/L). Three isolates (0.5%) had elevated MICs for vancomycin (4-8 mg/L); however, these isolates originated from the same patient, who was receiving long-term intravenous vancomycin treatment. High-level clindamycin resistant isolates (MIC >256 mg/L) peaked in 1997 with 39 of 95 (41%) and out of these, 36% were also highly resistant to erythromycin. beta-Lactams such as penicillin V and piperacillin displayed MIC(90)s of 8 and 32 mg/L, respectively, whereas MICs of cefuroxime were >256 mg/L for all isolates. Universal resistance to ciprofloxacin and levofloxacin was found, and resistance to moxifloxacin increased from 4% of isolates in 2004 to 23% in 2007. Notably, these moxifloxacin-resistant isolates did not belong to the recent epidemic PCR ribotype 027, but to the pre-existing epidemic type 012 (82%), and these isolates accounted for the majority of isolates that were resistant to clindamycin (70%), tetracycline (84%) and rifampicin (92%) as well. This investigation of susceptibility data on clinical C. difficile isolates showed variations of multiresistance to be due to a specific PCR ribotype 012, emphasizing the importance of genotyping when evaluating emerging resistance over time.

Update of Clostridium difficile infection due to PCR ribotype 027 in Europe, 2008.

Outbreaks of Clostridium difficile infections (CDI) with increased severity, high relapse rate and significant mortality have been related to the emergence of a new, hypervirulent C. difficile strain in North America and Europe. This emerging strain is referred to as PCR ribotype 027 (Type 027). Since 2005, individual countries have developed surveillance studies about the spread of type 027.C. difficile Type 027 has been reported in 16 European countries. It has been responsible for outbreaks in Belgium, Germany, Finland, France, Ireland, Luxembourg, The Netherlands, Switzerland and the United Kingdom (England, Wales, Northern Ireland and Scotland). It has also been detected in Austria, Denmark, Sweden, Norway, Hungary, Poland and Spain. Three countries experienced imported patients with CDI due to Type 027 who acquired the infection abroad.The antimicrobial resistance pattern is changing, and outbreaks due to clindamycin-resistant ermB positive Type 027 strains have occurred in three European countries. Ongoing epidemiological surveillance of cases of CDI, with periodic characterisation of the strains involved, is required to detect clustering of cases in time and space and to monitor the emergence of new, highly virulent clones.

Update of Clostridium difficile-associated disease due to PCR ribotype 027 in Europe.

Recent outbreaks of Clostridium difficile-associated diarrhoea (CDAD) with increased severity, high relapse rate and significant mortality have been related to the emergence of a new, hypervirulent C. difficile strain in North America, Japan and Europe. Definitions have been proposed by the European Centre of Disease Prevention and Control (ECDC) to identify severe cases of CDAD and to differentiate community-acquired cases from nosocomial CDAD (http://www.ecdc.europa.eu/documents/pdf/Cl_dif_v2.pdf). CDAD is mainly known as a healthcare-associated disease, but it is also increasingly recognised as a community-associated disease. The emerging strain is referred to as North American pulsed-field type 1 (NAP1) and PCR ribotype 027. Since 2005, individual countries have developed surveillance studies to monitor the spread of this strain. C. difficile type 027 has caused outbreaks in England and Wales, Ireland, the Netherlands, Belgium, Luxembourg, and France, and has also been detected in Austria, Scotland, Switzerland, Poland and Denmark. Preliminary data indicated that type 027 was already present in historical isolates collected in Sweden between 1997 and 2001.

Mutations in fusA associated with posttherapy fusidic acid resistance in Clostridium difficile.

In silico, we identified fusA (2,067 bp) in Clostridium difficile 630. Sequencing of fusA in posttherapy fusidic acid-resistant C. difficile isolates from 12 patients with C. difficile-associated diarrhea (CDAD) identified fusA mutations, one or two nonsynonymous substitutions, or in one case a deletion of one codon associated with resistance. Five of these mutations have previously been described in fusA of fusidic acid-resistant Staphylococcus aureus, but seven were novel fusA mutations. Fusidic acid monotherapy for CDAD seemed to rapidly select conserved resistant mutants.

Molecular epidemiology of hospital-associated and community-acquired Clostridium difficile infection in a Swedish county.

All episodes of Clostridium difficile associated diarrhea (CDAD) diagnosed in a defined population of 274,000 including one tertiary and two primary hospitals and their catchment areas were studied during 12 months. The annual CDAD incidence in the county was 97 primary episodes per 100,000, and 78% of all episodes were classified as hospital associated with a mean incidence of 5.3 (range, 1.4 to 6.5) primary episodes per 1,000 admissions. The incidence among hospitalized individuals was 1,300-fold higher than that in the community (33,700 versus 25 primary episodes per 100,000 persons per year), reflecting a 37-fold difference in antibiotic consumption (477 versus 13 defined daily doses [DDD]/1,000 persons/day) and other risk factors. Three tertiary hospital wards with the highest incidence (13 to 36 per 1,000) had CDAD patients of high age (median age of 80 years versus 70 years for other wards, P < 0.001), long hospital stay (up to 25 days versus 4 days), or a high antibiotic consumption rate (up to 2,427 versus 421 DDD/1,000 bed days). PCR ribotyping of C. difficile isolates available from 330 of 372 CDAD episodes indicated nosocomial acquisition of the strain in 17 to 27% of hospital-associated cases, depending on the time interval between index and secondary cases allowed (2 months or up to 12 months), and only 10% of recurrences were due to a new strain of C. difficile (apparent reinfection). In other words, most primary and recurring episodes were apparently caused by the patient's endogenous strain rather than by one of hospital origin. Typing also indicated that a majority of C. difficile strains belonged to international serotypes, and the distribution of types was similar within and outside hospitals and in primary and relapsing CDAD. However, type SE17 was an exception, comprising 22% of hospital isolates compared to 6% of community isolates (P = 0.008) and causing many minor clusters and a silent nosocomial outbreak including 36 to 44% of the CDAD episodes in the three high-incidence wards.

Comparison of AP-PCR typing and PCR-ribotyping for estimation of nosocomial transmission of Clostridium difficile.

We recently attempted to clarify an increased incidence of Clostridium difficile-associated diarrhoea (CDAD) in our hospital by arbitrarily primed polymerase chain reaction (AP-PCR) typing of isolates from 147 consecutive patients collected during a 12 month period (Wullt et al. J Hosp Infect 1999;43:265-273). In the present study we compared the results based on previous AP-PCR data with those based on recent PCR ribotyping of the same isolates and re-analysis of a subset of isolates by AP-PCR typing. The pattern of PCR ribotypes was similar among inpatients and outpatients. A cluster of three closely related PCR ribotypes, related to those of the serogroup H and A8 type strains, dominated and comprised 31% of inpatient and 28% of outpatient C. difficile isolates. The apparent nosocomial transmission rate among inpatients with CDAD was only 9% by AP-PCR typing compared with 18 or 36% by PCR ribotyping depending on the definition used (proportion of patients sharing C. difficile type and ward within two or 12 months). Corresponding rates for all CDAD patients were 5% by AP-PCR and 11 or 21% by PCR ribotyping. Thus, most CDAD patients apparently became ill due to their endogenous strain of C. difficile. Because of the low concordance between the two typing methods the proportion of patients fulfilling the criteria for nosocomial transmission by both methods was only 1%. Re-examination of isolates from patients with recurrences revealed a reproducibility problem with AP-PCR typing. We conclude, that of these two PCR-based options for typing of C. difficile PCR ribotyping offers a superior experimental robustness compared with AP-PCR typing.

Dynamics of penicillin-susceptible clones in invasive pneumococcal disease.

In a 10-year period, 1987-1997, there was a >4-fold increase in the rate of pneumococcal bacteremia in Sweden. Invasive pneumococcal isolates (n=1136), which were obtained from 18 Swedish clinical microbiology laboratories from 1987 through 1997, and other national and international isolates were serotyped, and their clonal relationships were determined by molecular typing. The increase in invasive pneumococcal disease in Sweden during this period was associated particularly with an increase in isolates of serotypes 1 and 14. A 3-fold increase of type 14 was seen from 1987 through 1992, and a 10-fold increase of type 1 occurred from 1992 through 1997. One dominating penicillin-susceptible clone of type 14 was responsible for the increase of type 14 during the first 5 years. This clone also was found in Canada and the United States and was shown by multilocus sequence typing to correspond to a previously identified hyper-virulent clone. A novel penicillin-susceptible clone of type 1, which was not found among invasive isolates from 1987 or 1992, was responsible for the increase of serotype 1 during the last 5 years. These results illustrate the ability of virulent penicillin-susceptible pneumococcal clones to emerge and spread rapidly within a country.

Toxins, butyric acid, and other short-chain fatty acids are coordinately expressed and down-regulated by cysteine in Clostridium difficile.

It was recently found that a mixture of nine amino acids down-regulate Clostridium difficile toxin production when added to peptone yeast extract (PY) cultures of strain VPI 10463 (S. Karlsson, L. G. Burman, and T. Akerlund, Microbiology 145:1683-1693, 1999). In the present study, seven of these amino acids were found to exhibit a moderate suppression of toxin production, whereas proline and particularly cysteine had the greatest impact, on both reference strains (n = 6) and clinical isolates (n = 28) of C. difficile (>99% suppression by cysteine in the highest toxin-producing strain). Also, cysteine derivatives such as acetylcysteine, glutathione, and cystine effectively down-regulated toxin expression. An impact of both cysteine and cystine but not of thioglycolate on toxin yield indicated that toxin expression was not regulated by the oxidation-reduction potential. Several metabolic pathways, including butyric acid and butanol production, were coinduced with the toxins in PY and down-regulated by cysteine. The enzyme 3-hydroxybutyryl coenzyme A dehydrogenase, a key enzyme in solventogenesis in Clostridium acetobutylicum, was among the most up-regulated proteins during high toxin production. The addition of butyric acid to various growth media induced toxin production, whereas the addition of butanol had the opposite effect. The results indicate a coupling between specific metabolic processes and toxin expression in C. difficile and that certain amino acids can alter these pathways coordinately. We speculate that down-regulation of toxin production by the administration of such amino acids to the colon may become a novel approach to prophylaxis and therapy for C. difficile-associated diarrhea.

Clustering of clinical strains of Helicobacter pylori analyzed by two-dimensional gel electrophoresis.

Strain variations of Helicobacter pylori have been tested by numerous methods and compared among different patient groups. The aim of this study was to investigate whether H. pylori expresses disease-specific proteins that can be detected by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). H. pylori strains isolated from duodenal ulcer, gastric cancer, and gastritis patients were analyzed. Extensive variation in spot patterns was observed between the strains, but a dendrogram analysis revealed that some strains within each disease group clustered together. Eight proteins were sequenced and found in the H. pylori genome sequence. 2-D PAGE is a useful method for studies of protein expression and for highlighting the extensive strain variation that H. pylori exhibits.

On the origin of branches in Escherichia coli.

Some Escherichia coli strains with impaired cell division form branched cells at high frequencies during certain growth conditions. Here, we show that neither FtsI nor FtsZ activity is required for the development of branches. Buds did not form at specific positions along the cell surface during high-branching conditions. Antibiotics affecting cell wall synthesis had a positive effect on branch formation in the case of ampicillin, cephalexin, and penicillin G, whereas mecillinam and D-cycloserine had no substantial effect. Altering the cell morphology by nutritional shifts showed that changes in morphology preceded branching, indicating that the cell's physiological state rather than specific medium components induced branching. Finally, there was no increased probability for bud formation in the daughters of a cell with a bud or branch, showing that bud formation is a random event. We suggest that branch formation is caused by abnormalities in cell wall elongation rather than by aberrant cell division events.

Analysis of protein synthesis rates after initiation of chromosome replication in Escherichia coli.

The aim of this study was to investigate whether the synthesis rates of some proteins change after the initiation of replication in Escherichia coli. An intR1 strain, in which chromosome replication is under the control of an R1 replicon integrated into an inactivated oriC, was used to synchronize chromosome replication, and the rates of protein synthesis were analyzed by two-dimensional polyacrylamide gel electrophoresis of pulse-labeled proteins. Computerized image analysis was used to search for proteins whose expression levels changed at least threefold after initiation of a single round of chromosome replication, which revealed 7 out of about 1,000 detected proteins. The various synthesis rates of three of these proteins turned out to be caused by unbalanced growth and the synthesis of one protein was suppressed in the intR1 strain. The rates of synthesis of the remaining three could be correlated only to the synchronous initiation of replication. These three proteins were analyzed by peptide mass mapping and appeared to be the products of the dps, gapA, and pyrI genes. Thus, the expression of the vast majority of proteins is not influenced by the state of chromosome replication, and a possible role of the replication-associated expression changes of the three identified proteins in the cell cycle is not clear.

Analysis of cell size and DNA content in exponentially growing and stationary-phase batch cultures of Escherichia coli.

Escherichia coli strains were grown in batch cultures in different media, and cell size and DNA content were analyzed by flow cytometry. Steady-state growth required large dilutions and incubation for many generations at low cell concentrations. In rich media, both cell size and DNA content started to decrease at low cell concentrations, long before the cultures left the exponential growth phase. Stationary-phase cultures contained cells with several chromosomes, even after many days, and stationary-phase populations exclusively composed of cells with a single chromosome were never observed, regardless of growth medium. The cells usually contained only one nucleoid, as visualized by phase and fluorescence microscopy. The results have implications for the use of batch cultures to study steady-state and balanced growth and to determine mutation and recombination frequencies in stationary phase.

Inhibition and restart of initiation of chromosome replication: effects on exponentially growing Escherichia coli cells.

Escherichia coli strains in which initiation of chromosome replication could be specifically blocked while other cellular processes continued uninhibited were constructed. Inhibition of replication resulted in a reduced growth rate and in inhibition of cell division after a time period roughly corresponding to the sum of the lengths of the C and D periods. The division inhibition was not mediated by the SOS regulon. The cells became elongated, and a majority contained a centrally located nucleoid with a fully replicated chromosome. The replication block was reversible, and restart of chromosome replication allowed cell division and rapid growth to resume after a time delay. After the resumption, the septum positions were nonrandomly distributed along the length axis of the cells, and a majority of the divisions resulted in at least one newborn cell of normal size and DNA content. With a transient temperature shift, a single synchronous round of chromosome replication and cell division could be induced in the population, making the constructed system useful for studies of cell cycle-specific events. The coordination between chromosome replication, nucleoid segregation, and cell division in E. coli is discussed.

Branched Escherichia coli cells.

We report that the normally rod-shaped bacterium Escherichia coli can form branched cells. These were found in strains in which chromosome replication or nucleoid segregation was disturbed, e.g. in minB mutants, intR1 strains, and in strains exhibiting stable DNA replication. Often chromosome DNA was found to be located in the branch point of the cells. The branching frequency was dependent upon the growth medium: in rich medium no branched cells were found, whereas in minimal medium containing acetate and casamino acids the frequency of branched cells was increased. The genetic background of the strains also affected the tendency to branch. Furthermore, electron microscopy of thin-sectioned branched cells revealed additional membrane-like structures, which were not observed in wild-type cells. Finally, the branched cells are compared with bacteria that normally branch, and probable causes for branching in E. coli are discussed.

Cell division in Escherichia coli minB mutants.

In Escherichia coli minB mutants, cell division can take place at the cell poles as well as non-polarly in the cell. We have examined growth, division patterns, and nucleoid distribution in individual cells of a minC point mutant and a minB deletion mutant, and compared them to the corresponding wild-type strain and an intR1 strain in which the chromosome is over-replicated. The main findings were as follows. In the minB mutants, polar and non-polar divisions appeared to occur independently of each other. Furthermore, the timing of cell division in the cell cycle was found to be severely affected. In addition, nucleoid conformation and distribution were considerably disturbed. The results obtained call for a re-evaluation of the role of the MinB system in the E. coli cell cycle, and of the concept that limiting quanta of cell division factors are regularly produced during the cell cycle.