Monica Österblad

Antibiotic resistance: How wild are wild mammals?

In bacteria associated with humans, antimicrobial resistance is common, both in clinical isolates and in the less-studied commensal flora, and it is thought that commensal and environmental bacteria might be a hidden reservoir of resistance. Gilliver et al. have reported that resistance is also prevalent in faecal bacteria from wild rodents living in northwest England. Here we test the faeces of moose, deer and vole in Finland and find an almost complete absence of resistance in enterobacteria. Resistance is thus not a universal property of enterobacterial populations, but may be a result of the human use of antibiotics.

A between-Species Comparison of Antimicrobial Resistance in Enterobacteria in Fecal Flora

ABSTRACT Enterobacteria in fecal flora are often reported to be highly resistant. Escherichia coli is the main species; resistance data on other species are rare. To assess the effect of the hosts environment, antimicrobial resistance was determined in fecal species of the family Enterobacteriaceae from three populations: healthy people (HP)(n = 125) with no exposure to antimicrobials for 3 months preceding sampling, university hospital patients (UP) (n = 159) from wards where the antibiotic use was 112 defined daily doses (DDD)/bed/month, and geriatric long-term patients (LTP) (n = 74) who used 1.8 DDD/bed/month. The mean length of hospital stay was 5 days for the UP and 22 months for the LTP. The isolates were identified to at least genus level, and MICs of 16 antimicrobials were determined. From the university hospital, resistance data on clinicalEnterobacteriaceae isolates were also collected. Resistance data for on average two different isolates per sample (range, 1 to 5) were analyzed: 471 E. coli isolates and 261 otherEnterobacteriaceae spp. Resistance was mainly found amongE. coli; even in HP, 18% of E. coli isolates were resistant to two or more antimicrobial groups, with MIC patterns indicative of transferable resistance. Other fecal enterobacteria were generally susceptible, with little typically transferable multiresistance. Clinical Klebsiella andEnterobacter isolates were significantly more resistant than fecal isolates. The resistance patterns at both hospitals mirrored the patterns of antibiotic use, but LTP E. coli isolates were significantly more resistant than those from UP. Conditions permitting an efficient spread may have been more important in sustaining high resistance levels in the LTP. E. coli was the main carrier of antimicrobial resistance in fecal flora; resistance in other species was rare in the absence of antimicrobial selection.

Carbapenemase-producing Enterobacteriaceae in Finland: the first years (2008–11)

OBJECTIVES Carbapenemase-producing Enterobacteriaceae (CPE) are becoming a global problem; they are often resistant to nearly all available antibiotics. Here we report details on all Finnish CPE isolates found until the end of 2011: carbapenemase genes, travel history and multilocus sequence typing (MLST) data. METHODS Enterobacteriaceae sent to the Antimicrobial Resistance Unit of the National Institute for Health and Welfare were tested for susceptibility to carbapenems, screened for carbapenemases by PCR and isolates with decreased susceptibility to carbapenems were tested for hydrolysis of imipenem. Carbapenemase-producing Escherichia coli and Klebsiella pneumoniae isolates were typed by MLST. RESULTS In all, 26 CPE strains were found from 25 patients: 10 with OXA-48-like enzymes, 5 with KPC, 4 with VIM, 3 with NDM, 3 with IMI/NMC-A and 1 with GES-14. The species were K. pneumoniae (n = 16), E. coli (n = 6), Enterobacter cloacae (n = 3) and Raoultella planticola (n = 1). Of the 25 patients, 18 had a known travel history/hospital transfer from abroad. Local spread/transmission was suspected in 2011, but there were no hospital outbreaks. The K. pneumoniae multilocus sequence types ST258, ST182, ST147, ST244, ST14, ST13, ST383, ST101 and ST15, and the E. coli sequence types ST38 and ST90 were found. Many of these are global epidemic clones. CONCLUSIONS CPE strains are increasingly found in Finland, but still at a very low prevalence.

Evaluation of the Carba NP Test for Carbapenemase Detection

ABSTRACT The Carba NP test was evaluated against a panel of 61 carbapenemase-producing bacterial species (15 producing class A carbapenemases, 15 producing class D carbapenemases, and 31 producing metallo-β-lactamases) and against 111 isolates with non-wild-type carbapenem susceptibility but not producing carbapenemase. Carbapenemase production was verified by PCR and UV-spectrophotometric measurement of imipenem hydrolysis. No false positives were seen, but there were consistent problems with the detection of OXA-48-like enzymes and also some rarer class A enzymes.

Detection and molecular genetics of extended-spectrum beta-lactamases among cefuroxime-resistant Escherichia coli and Klebsiella spp. isolates from Finland, 2002–2004

Extended-spectrum beta-lactamase (ESBL) producing Escherichia coli and Klebsiella spp. isolates are spreading and becoming an increasing problem concerning treatment, diagnostics and hospital hygiene. We wanted to discover which genotypes are occurring in Finland and to assess the CLSI screening method. The isolates were collected from 26 laboratories during a 3-y period from 2002 to 2004. We studied the zone diameters by disk diffusion according to CLSI recommendations. ESBL genes were detected by PCR and the TEM and SHV genes were sequenced traditionally, while the CTX-M isolates were analysed with pyrosequencing. Of the 402 isolates included in the study, 269 (67%) were confirmed to be ESBL producers according to the CLSI criteria. The CTX-M genes were the most prevalent, especially the combination of a CTX-M-1-group and a TEM-1 gene. In our material there were few isolates that had an ESBL gene but were negative in the CLSI ESBL confirmatory test. During recent y especially the CTX-M producing isolates have increased in Europe and now they are also found in Finland with increasing prevalence.

Antimicrobial and mercury resistance in aerobic gram-negative bacilli in fecal flora among persons with and without dental amalgam fillings.

Antimicrobial resistance is more widespread than can be accounted for as being a consequence of the selection pressure caused by the use of antibiotics alone. In this study, we tested the hypothesis that a high mercury content in feces might select for mercury-resistant bacteria and thus for antimicrobial resistance linked to mercury resistance. Three subject groups with different exposures to dental amalgam fillings were compared. None of the subjects had taken antimicrobial agents during the three preceding months or longer. The group exposed to dental amalgam (n = 92) had 13 times more mercury in feces than the group that had never been exposed to amalgam (n = 43) and the group whose amalgam fillings had been removed (n = 56). No significant differences in either mercury resistance or antibiotic resistance in the fecal aerobic gram-negative flora of these subject groups were seen. The following antimicrobial resistance frequencies were detected with a replica plating method: > or = 1% resistance was seen in 40% of the subjects for ampicillin, 14% of the subjects for cefuroxime, 6% of the subjects for nalidixic acid, 14% of the subjects for trimethoprim, 19% of the subjects for sulfamethoxazole, and 25% of the subjects for tetracycline. The amount of mercury in feces derived from amalgam was not selective for any resistance factors in aerobic gram-negative bacteria, but antimicrobial resistance was widespread even among healthy subjects with no recent exposure to antibiotics.

Resistance to Mercury and Antimicrobial Agents in Streptococcus mutans Isolates from Human Subjects in Relation to Exposure to Dental Amalgam Fillings

ABSTRACT Resistance to cefuroxime, penicillin, tetracycline, and mercury is reported for 839 Streptococcus mutans isolates from 209 human study subjects. The MICs of these drugs did not differ for isolates from one dental amalgam group and two nonamalgam subsets: a group with no known exposure to amalgam and a group whose members had their amalgam fillings removed.

Evaluation of a New Cellulose Sponge-Tipped Swab for Microbiological Sampling: a Laboratory and Clinical Investigation

ABSTRACT A new type of swab (Cellswab; Cellomeda, Turku, Finland), utilizing a highly absorbent cellulose viscose sponge material, was compared to some traditional swabs. The survival of 14 aerobic and 10 anaerobic and microaerophilic bacterial species in the Cellswab, two commercial swab transport systems (Copan, Brescia, Italy, and Orion Diagnostica, Espoo, Finland), and one Dacron swab (Technical Service Consultants Ltd. [TSC], Heywood, United Kingdom) was evaluated. Bacteria were suspended in broth, into which the swabs were dipped. The Cellswab absorbed 1.3 times more fluid and released 3.5 times more fluid upon plating than the other swabs. Aerobic bacteria were stored in dry tubes, the others in transport medium, at 4°C and room temperature (RT), for up to 14 days. Swab samples were transferred to plates at 0, 1, 2, 4, 7, and 14 days. For 10 strains the Cellswab yielded ≥10% of the original CFU for longer than all the other swabs. In the clinical study, the ability of the Cellswab to detect beta-hemolytic streptococci from throat samples (n = 995) was compared to that of the TSC Dacron swab. The swabs performed equally, both when their samples were transferred to plates immediately and after storage for 1 day at 4°C or RT. The changes in normal microbiota after storage were also similar. The Cellswab was found to perform at least as well as ordinary swabs. It was better at storing fastidious strains, and at keeping bacteria viable for long storage times; it might well be a useful replacement or complement to ordinary swabs.

Long-term antimicrobial resistance in Escherichia coli from human intestinal microbiota after administration of clindamycin

The aim of this study was to gain better knowledge of how the intestinal microbiota are affected over time after administration of an antimicrobial agent. This study monitored the prevalence and frequencies of antibiotic resistance in Enterobacteriaceae against 17 antimicrobial agents, during a 2-y period, in consecutive faecal samples collected from 8 healthy volunteers. Four subjects had received 150 mg clindamycin perorally for 7 d, while 4 non-exposed subjects served as a control group. The samples from both groups were cultured and screened for Enterobacteriaceae. The highest incidence of resistance observed was to ampicillin. The ampicillin resistance is due to production of the beta-lactamase TEM-1. The administration of clindamycin had a prolonged impact on the composition of the microbiota, even though enterobacteria are intrinsically resistant to clindamycin; the level of resistance in Escherichia coli isolates was elevated after administration and persisted up to 9 months after administration. After 9 months the susceptibility levels in the exposed group were similar to those at d 0.

Evaluation of two commercial carbapenemase gene assays, the Rapidec Carba NP test and the in-house Rapid Carba NP test, on bacterial cultures

Sir, Carbapenemase-producing organisms (CPO) are recognized as one of the biggest problems in infectious diseases today: how do you treat a patient infected with an organism that might no longer be eliminated by antibiotics? Not only do you have to produce susceptibility results, but knowing the mechanism behind the resistance is also crucial, e.g. many of the new antibiotic/inhibitor combinations are effective only against class A enzymes. With a long list of enzymes and a broad spectrum of susceptibility phenotypes, this is a big challenge for clinical laboratories. We need methods that are simple to set up and perform, have a low rate of false negatives and are reasonably specific. We compared two commercial molecular assays [Check-Direct CPE (Check-Points Health, Wageningen, the Netherlands) and Xpert Carba-R (Cepheid, Maurens-Scopont, France)] and two phenotypic tests [the in-house Rapid Carba NP test and the commercially available version, i.e. Rapidec Carba NP (bioMérieux, Marcy-l’Étoile, France)] using a panel of bacteria with known carbapenemase status, selected among strains sent to our reference laboratory during 2008 to March 2014 from all Finnish clinical laboratories. Of the 94 isolates tested, 57 were CPO (Table 1): 45 Enterobacteriaceae (11 KPC, 15 NDM, 9 OXA-48, 5 OXA-181 and 5 VIM), 9 Pseudomonas aeruginosa (6 VIM and 3 IMP) and 3 Acinetobacter spp. (2 NDM and 1 VIM). They had been confirmed with our in-house PCR (testing for KPC, NDM, OXA-48-like, VIM, IMP, GES and SPM) and UV-spectrophotometric imipenem hydrolysis assay. Of the non-CPO, all had been referred to us because of non-WT susceptibility to carbapenems (screening cut-off for Enterobacteriaceae, zone diameters: meropenem ,22 mm, imipenem ,21 mm, ertapenem ,25 mm; and screening cut-off for Pseudomonas, zone diameters: meropenem ,24 mm and imipenem ,20 mm); 11 Enterobacteriaceae isolates had ambiguous (borderline) results in the hydrolysis assay, but no carbapenemase genes were found with the in-house PCR; the borderline result was assumed to be caused by increased chromosomal AmpC enzyme activity. Whole-genome sequences of two similar strains have since been studied and only chromosomal genes found. Nineteen of the non-CPO Enterobacteriaceae had ESBL or transferable ampC genes (tested for CTX-M, SHV, TEM, CMY, DHA, ACC, FOX, MOX and EBC), seven P. aeruginosa and one Klebsiella pneumoniae strains had reduced susceptibility to carbapenems but no carbapenemase activity or transferable b-lactamase genes. Check-Direct CPE was performed as outlined in the manufacturer’s manual and run on a LightCycler 480 II (Roche, Basel, Switzerland). Xpert Carba-R was performed using the manufacturer’s standard protocol and run on a GeneXpert IV. False negative results were retested once. In-house Carba NP was conducted using the Rapid protocol, as taught to the participants of the Capacity-Building Workshop of the ECDC EuSCAPE project. The Rapidec Carba NP test was done according to the kit instructions, without modifications. For both phenotypic tests, strains were grown on Mueller–Hinton-II agar overnight and results were read after 2 h. The performers and interpreters of all the assays were blinded to the properties of the isolates and each Carba NP assay was independently interpreted by two technicians. No false positives were seen with the first three assays (Table 1), but the Rapidec Carba NP test surprisingly produced 14 false positives. On the other hand, there were only 4 false negatives among the OXA-48-like positive isolates, compared with 12 with the in-house protocol. Others have also experienced difficulties with OXA-48 and seen false positives with the Rapidec Carba NP test. The reason we observed this many false positives is probably due to our negative panel, which was not random, but hand-picked to be extra challenging (most had some kind of b-lactamase activity, not defined as carbapenemase). The species and mechanisms of the false positives were more or less a cross-section of the negative panel: eight ESBL positive (1 ESBL-SHV Escherichia coli, 7 CTX-M: 2 K. pneumoniae, 5 E. coli), one CMY-positive Proteus mirabilis, two Enterobacter cloacae and one Klebsiella oxytoca with borderline results in the hydrolysis assay, and the K. pneumoniae without transferable genes and negative hydrolysis (ertapenem disc 23 mm). Interpretation was also more difficult with the commercial kit, but interpreting orange-red wells as negative, to get fewer false positives, would have given more false negatives. pH might be the fundamental problem with OXA-48-like enzymes: Studentova et al. showed that bicarbonate added to test media significantly enhanced enzyme activity, i.e. a drop in pH, such as happens in a positive Carba NP test, can act to inhibit OXA-48. The two technicians had the same Carba NP results for all but one OXA-181-carrying isolate; this was interpreted in favour of the positive result. In-house Carba NP surprisingly did not detect one KPC isolate and we found no explanation for this: the strain was a non-mucous ST258 K. pneumoniae of Greek origin that was fully resistant (meropenem MIC, 8 mg/L; imipenem MIC, 16 mg/L). It was also weakly positive with the Rapidec Carba NP test. Hands-on time with Xpert Carba-R was much less than with the other methods and it allowed testing directly from a transport swab and also produced the fastest results (,1 h). Check-Direct CPE required isolated DNA and took 2.5 h. The Rapidec Carba NP test took up to 2 h and 40 min from colony to result; the in-house version took slightly longer, depending on how far in advance the reagents had been prepared.