Lene Nørby Nielsen

Review and phylogenetic analysis of qac genes that reduce susceptibility to quaternary ammonium compounds in Staphylococcus species

The qac genes of Staphylococcus species encode multidrug efflux pumps: membrane proteins that export toxic molecules and thus increase tolerance to a variety of compounds such as disinfecting agents, including quaternary ammonium compounds (for which they are named), intercalating dyes and some antibiotics. In Stapylococcus species, six different plasmid-encoded Qac efflux pumps have been described, and they belong to two major protein families. QacA and QacB are members of the Major Facilitator Superfamily, while QacC, QacG, QacH, and QacJ all belong to the Small Multidrug Resistance (SMR) family. Not all SMR proteins are called Qac and the reverse is also true, which has caused confusion in the literature and in gene annotations. The discovery of qac genes and their presence in various staphylococcal populations is briefly reviewed. A sequence comparison revealed that some of the PCR primers described in the literature for qac detection may miss particular qac genes due to lack of DNA conservation. Despite their resemblance in substrate specificity, the Qac proteins belonging to the two protein families have little in common. QacA and QacB are highly conserved in Staphylococcus species, while qacA was also detected in Enterococcus faecalis, suggesting that these plasmid-born genes have spread across bacterial genera. Nevertheless, these qacA and qacB genes are quite dissimilar to their closest homologues in other organisms. In contrast, SMR-type Qac proteins display considerable sequence variation, despite their short length, even within the Staphylococcus genus. Phylogenetic analysis of these genes identified similarity to a large number of other SMR members, found in staphylococci as well as in other genera. A number of phylogenetic trees of SMR Qac proteins are presented here, starting with genes present in S. aureus and S. epidermidis, and extending this to related genes found in other species of this genus, and finally to genes found in other genera.

MLST clustering of Campylobacter jejuni isolates from patients with gastroenteritis, reactive arthritis and Guillain-Barré syndrome

Aims:  To determine the diversity and population structure of Campylobacter jejuni (C. jejuni) isolates from Danish patients and to examine the association between multilocus sequence typing types and different clinical symptoms including gastroenteritis (GI), Guillain–Barré syndrome (GBS) and reactive arthritis (RA).

Recently introduced qacA/B genes in Staphylococcus epidermidis do not increase chlorhexidine MIC/MBC

OBJECTIVES Chlorhexidine is used as a disinfectant to prevent surgical infections. Recently, studies have indicated that chlorhexidine usage has selected methicillin-resistant Staphylococcus aureus strains that are tolerant to chlorhexidine and that this may be related to the presence of the qacA/B-encoded efflux pumps. Here, we evaluated if high-level exposure to chlorhexidine selects for tolerant colonizing Staphylococcus epidermidis and we addressed the consequences of long-term exposure to chlorhexidine. METHODS Chlorhexidine susceptibility and carriage of qacA/B was determined for colonizing S. epidermidis isolated from scrub nurses heavily exposed to chlorhexidine and were compared with isolates from non-users of chlorhexidine hand rubs. S. epidermidis blood isolates from the 1960s, before the wider introduction of chlorhexidine to the market, were also tested and compared with recently collected S. epidermidis blood isolates. RESULTS There was no correlation between the use of chlorhexidine in scrub nurses and the presence of qacA/B genes in S. epidermidis isolates or increased MICs/MBCs of chlorhexidine for S. epidermidis isolates. While 55% of current blood isolates harboured the qacA/B genes, none of the 33 historical S. epidermidis isolates did, although their MICs and MBCs of chlorhexidine were comparable to those for current isolates. CONCLUSIONS Chlorhexidine used as a hand rub does not select for S. epidermidis isolates with increased MICs or MBCs of chlorhexidine. However, the absence of qacA/B genes in S. epidermidis isolates obtained in the 1960s suggests that long-term use of biocides like chlorhexidine or related compounds may select for the presence of qacA/B genes.

Staphylococcus epidermidis Isolated in 1965 Are More Susceptible to Triclosan than Current Isolates

Since its introduction to the market in the 1970s, the synthetic biocide triclosan has had widespread use in household and medical products. Although decreased triclosan susceptibility has been observed for several bacterial species, when exposed under laboratory settings, no in vivo studies have associated triclosan use with decreased triclosan susceptibility or cross-resistance to antibiotics. One major challenge of such studies is the lack of strains that with certainty have not been exposed to triclosan. Here we have overcome this challenge by comparing current isolates of the human opportunistic pathogen Staphylococcus epidermidis with isolates collected in the 1960s prior to introduction of triclosan to the market. Of 64 current S. epidermidis isolates 12.5% were found to have tolerance towards triclosan defined as MIC≥0.25 mg/l compared to none of 34 isolates obtained in the 1960s. When passaged in the laboratory in the presence of triclosan, old and current susceptible isolates could be adapted to the same triclosan MIC level as found in current tolerant isolates. DNA sequence analysis revealed that laboratory-adapted strains carried mutations in fabI encoding the enoyl-acyl carrier protein reductase isoform, FabI, that is the target of triclosan, and the expression of fabI was also increased. However, the majority of the tolerant current isolates carried no mutations in fabI or the putative promoter region. Thus, this study indicates that the widespread use of triclosan has resulted in the occurrence of S. epidermidis with tolerance towards triclosan and that the adaptation involves FabI as well as other factors. We suggest increased caution in the general application of triclosan as triclosan has not shown efficacy in reducing infections and is toxic to aquatic organisms.

Diverse modulation of spa transcription by cell wall active antibiotics in Staphylococcus aureus

BackgroundThe aim of this study was to investigate the effect of various classes of clinically relevant antibiotics at sub-lethal concentrations on virulence gene expression and biofilm formation in Staphylococcus aureus.FindingsLacZ promoter fusions of genes related to staphylococcal virulence were used to monitor the effects of antibiotics on gene expression in a disc diffusion assay. The selected genes were hla and spa encoding α-hemolysin and Protein A, respectively and RNAIII, the effector molecule of the agr quorum sensing system. The results were confirmed by quantitative real-time PCR. Additionally, we monitored the effect of subinhibitory concentrations of antibiotics on the ability of S. aureus to form biofilm in a microtiter plate assay. The results show that sub-lethal antibiotic concentrations diversely modulate expression of RNAIII, hla and spa. Consistently, expression of all three genes were repressed by aminoglycosides and induced by fluoroquinolones and penicillins. In contrast, the β-lactam sub-group cephalosporins enhanced expression of RNAIII and hla but diversely affected expression of spa. The compounds cefalotin, cefamandole, cefoxitin, ceftazidime and cefixine were found to up-regulate spa, while down-regulation was observed for cefuroxime, cefotaxime and cefepime. Interestingly, biofilm assays demonstrated that the spa-inducing cefalotin resulted in less biofilm formation compared to the spa-repressing cefotaxime.ConclusionsWe find that independently of the cephalosporin generation, cephalosporins oppositely regulate spa expression and biofilm formation. Repression of spa expression correlates with the presence of a distinct methyloxime group while induction correlates with an acidic substituted oxime group. As cephalosporines target the cell wall penicillin binding proteins we speculate that subtle differences in this interaction fine-tunes spa expression independently of agr.

Prevalence, Antimicrobial Resistance and Risk Factors for Thermophilic Campylobacter Infections in Symptomatic and Asymptomatic Humans in Tanzania

The genus Campylobacter comprises members known to be a leading cause of foodborne gastrointestinal illness worldwide. A study was conducted to determine the epidemiology and antimicrobial resistance of Campylobacter in humans in Morogoro, Eastern Tanzania. Isolation of Campylobacter from stool specimens adopted the Cape Town protocol. Campylobacter isolates were preliminarily identified by conventional phenotypic tests and subsequently confirmed by matrix‐assisted laser desorption/ionization–time‐of‐flight (MALDI‐TOF) mass spectrometry and polymerase chain reaction. Antimicrobial resistance testing employed the disc diffusion method. A small proportion of the test isolates was also subjected to agar dilution method. Risk factors for human illness were determined in an unmatched case–control study. Thermophilic Campylobacter were isolated from 11.4% of the screened individuals (n = 1195). The agreement between PCR and MALDI‐TOF was perfect (κ = 1.0). Symptomatics and young individuals were infected with higher numbers than asymptomatic and adults, respectively. The majority (84.6%) of the isolates were C. jejuni and the remaining were C. coli. Isolates had highest resistance (95.6%) for colistin sulphate and lowest for ciprofloxacin (22.1%). The rates of resistance for other antibiotics (azithromycin, erythromycin, tetracycline, cephalothin, gentamycin, nalidixic acid, ampicillin, amoxycillin, norfloxacin, chloramphenicol) ranged from 44.1% to 89%. Comparison between disc diffusion and agar dilution methods indicated a good correlation, and the tests were in agreement to each other (κ ≥ 0.75). Human illness was found to be associated with young age and consumption of chicken meat and pre‐prepared salad. Our data indicate the presence of antibiotic‐resistant thermophilic Campylobacter in humans in the study area. There is a need for routine investigation of the presence of the organisms in gastroenteritis aetiology, including determination of their antibiotic susceptibilities.

Biocide Susceptibility of Staphylococcus aureus CC398 and CC30 Isolates from Pigs and Identification of the Biocide Resistance Genes, qacG and qacC.

OBJECTIVES Methicillin-resistant Staphylococcus aureus (MRSA), in particular clonal complex (CC) 398, is increasingly found in livestock. Recently, MRSA CC30 was identified in Danish pigs. We determined the susceptibility of porcine S. aureus isolates of CC398 and CC30 to disinfectants used in pig farming (benzalkonium chloride, hydrogen peroxide, formaldehyde, sodium hypochlorite, and caustic soda). Furthermore, efflux pump activity, antimicrobial resistance profiles, hemolysis properties, and the presence of toxic shock syndrome toxin-1 (TSST-1) and Panton-Valentine Leukocidin (PVL)-encoding virulence factors were investigated. METHODS Susceptibilities to biocides and antimicrobial agents of 79 porcine S. aureus isolates were determined by the microdilution method. Isolates comprised 21 methicillin-sensitive S. aureus (MSSA) and 40 MRSA isolates belonging to CC398 and 13 MSSA and 5 MRSA isolates belonging to CC30. The presence of quaternary ammonium compound (QAC) resistance efflux pumps was analyzed using an ethidium bromide accumulation assay. The presence of qac resistance genes in active efflux pump positive isolates was determined by whole-genome sequencing data. All isolates were screened for lukPV and tst genes with PCR, and hemolytic activities were determined using an agar plate assay. RESULTS S. aureus isolates did not show reduced susceptibility to the biocides tested. However, the QAC resistance gene, qacG, was detected in three MRSA CC30 isolates and the qacC in one MRSA CC30 isolate. CC30 isolates were generally more susceptible to non-beta-lactam antibiotics than CC398. Isolates generally had low hemolytic activity and none encoded PVL or TSST-1. CONCLUSION The presence of qac genes in European porcine S. aureus isolates and in livestock-associated MRSA CC30 is for the first time described in this study. This finding is concerning as it ultimately may compromise disinfection with QACs and thereby contribute to the selection and spread of MRSA CC30.

Novel screening assay for in vivo selection of Klebsiella pneumoniae genes promoting gastrointestinal colonisation

BackgroundKlebsiella pneumoniae is an important opportunistic pathogen causing pneumonia, sepsis and urinary tract infections. Colonisation of the gastrointestinal (GI) tract is a key step in the development of infections; yet the specific factors important for K. pneumoniae to colonize and reside in the GI tract of the host are largely unknown. To identify K. pneumoniae genes promoting GI colonisation, a novel genomic-library-based approach was employed.ResultsScreening of a K. pneumoniae C3091 genomic library, expressed in E. coli strain EPI100, in a mouse model of GI colonisation led to the positive selection of five clones containing genes promoting persistent colonisation of the mouse GI tract. These included genes encoding the global response regulator ArcA; GalET of the galactose operon; and a cluster of two putative membrane-associated proteins of unknown function. Both ArcA and GalET are known to be involved in metabolic pathways in Klebsiella but may have additional biological actions beneficial to the pathogen. In support of this, GalET was found to confer decreased bile salt sensitivity to EPI100.ConclusionsThe present work establishes the use of genomic-library-based in vivo screening assays as a valuable tool for identification and characterization of virulence factors in K. pneumoniae and other bacterial pathogens.

Commercial Biocides Induce Transfer of Prophage Φ13 from Human Strains of Staphylococcus aureus to Livestock CC398

Human strains of Staphylococcus aureus commonly carry the bacteriophage ΦSa3 that encodes immune evasion factors. Recently, this prophage has been found in livestock-associated, methicillin resistant S. aureus (MRSA) CC398 strains where it may promote human colonization. Here, we have addressed if exposure to biocidal products induces phage transfer, and find that during co-culture, Φ13 from strain 8325, belonging to ΦSa3 group, is induced and transferred from a human strain to LA-MRSA CC398 when exposed to sub-lethal concentrations of commercial biocides containing hydrogen peroxide. Integration of ΦSa3 in LA-MRSA CC398 occurs at multiple positions and the integration site influences the stability of the prophage. We did not observe integration in hlb encoding β-hemolysin that contains the preferred ΦSa3 attachment site in human strains, and we demonstrate that this is due to allelic variation in CC398 strains that disrupts the phage attachment site, but not the expression of β-hemolysin. Our results show that hydrogen peroxide present in biocidal products stimulate transfer of ΦSa3 from human to LA-MRSA CC398 strains and that in these strains prophage stability depends on the integration site. Knowledge of ΦSa3 transfer and stability between human and livestock strains may lead to new intervention measures directed at reducing human infection by LA-MRSA strains.

Exposure to a glyphosate-based herbicide formulation, but not glyphosate alone, has only minor effects on adult rat testis

Glyphosate has been suggested to be an endocrine disrupting chemical capable of disrupting male reproduction. There are conflicting data, however, with studies reporting effects from exposure to either glyphosate alone or to herbicide formulations, making comparisons difficult. We assessed rat testis histopathology and androgen function following two weeks exposure to either glyphosate at 2.5 and 25 mg/kg bw/day (5x and 50x Acceptable Daily Intake, ADI, respectively), or equivalent high dose of glyphosate in a herbicide formulation; Glyfonova. We observed no significant effects on testes or testosterone synthesis in rats exposed to glyphosate. Limited effects were observed in rats exposed to Glyfonova, with a small upregulation of the steroidogenic genes Cyp11a1 and Cyp17a1. We conclude that glyphosate alone has no effect on adult rat testis at exposure levels up to 25 mg/kg bw/day. Glyfonova induced only minor effects on steroidogenic gene expression, likely caused by additives other than glyphosate.