Characterization of Haemophilus influenzae Strains with Non-Enzymatic Resistance to β-Lactam Antibiotics Caused by Mutations in the PBP3 Gene in the Czech Republic in 2010–2018

The surveillance data on antibiotic resistance of Haemophilus influenzae have shown that strains with non-enzymatic resistance to β-lactam antibiotics have been on the rise in the Czech Republic over the last decade. This type of resistance is more difficult to detect than β-lactamase production. Analysis of 228 H. influenzae strains revealed that isolates with non-enzymatic resistance to β-lactams due to mutations in the ftsI gene could be reliably demonstrated by single run testing of susceptibility to amoxicillin/clavulanic acid (sensitivity of detection is 84.6%), cefuroxime (92.6%), ampicillin and penicillin (both 95.7%). Thirty-seven different amino acid substitution combinations were detected in the PBP3 protein at 23 positions (V329I, D350N, S357N, A368T, M377I, S385T, A388V, L389F, P393L, A437S, I449V, G490E, I491V, R501L, A502S, A502T, A502V, V511A, R517H, I519L, N526K, A530S, and T532S). The most common combination (35%) of amino acid substitutions was the combination D350N, M377I, A502V, N526K. Epidemiological typing does not indicate a clonal spread of a particular MLST type. Altogether there has been detected 74 STs. The most prevalent ST 1034 was associated mainly with a combination D350N, M377I, A502V, N526K. Clonal analysis revealed six clonal complexes (CCs) with the founder found, eight CCs without founder and 33 singletons.

Suitability of current typing procedures to identify epidemiologically linked human Giardia duodenalis isolates

Background Giardia duodenalis is a leading cause of gastroenteritis worldwide. Humans are mainly infected by two different subtypes, i.e., assemblage A and B. Genotyping is hampered by allelic sequence heterozygosity (ASH) mainly in assemblage B, and by occurrence of mixed infections. Here we assessed the suitability of current genotyping protocols of G. duodenalis for epidemiological applications such as molecular tracing of transmission chains. Methodology/Principal findings Two G. duodenalis isolate collections, from an outpatient tropical medicine clinic and from several primary care laboratories, were characterized by assemblage-specific qPCR (TIF, CATH gene loci) and a common multi locus sequence typing (MLST; TPI, BG, GDH gene loci). Assemblage A isolates were further typed at additional loci (HCMP22547, CID1, RHP26, HCMP6372, DIS3, NEK15411). Of 175/202 (86.6%) patients the G. duodenalis assemblage could be identified: Assemblages A 25/175 (14.3%), B 115/175 (65.7%) and A+B mixed 35/175 (20.0%). By incorporating allelic sequence heterozygosity in the analysis, the three marker MLST correctly identified 6/ 9 (66,7%) and 4/5 (80.0%) consecutive samples from chronic assemblage B infections in the two collections, respectively, and identified a cluster of five independent patients carrying assemblage B parasites of identical MLST type. Extended MLST for assemblage A altogether identified 5/6 (83,3%) consecutive samples from chronic assemblage A infections and 15 novel genotypes. Based on the observed A+B mixed infections it is estimated that only 75% and 50% of assemblage A or B only cases represent single strain infections, respectively. We demonstrate that typing results are consistent with this prediction. Conclusions/Significance Typing of assemblage A and B isolates with resolution for epidemiological applications is possible but requires separate genotyping protocols. The high frequency of multiple infections and their impact on typing results are findings with immediate consequences for result interpretation in this field.

Virulence Properties of mcr-1-Positive Escherichia coli Isolated from Retail Poultry Meat

The great plasticity and diversity of the Escherichia coli genome, together with the ubiquitous occurrence, make E. coli a bacterium of world-wide concern. Of particular interest are pathogenic strains and strains harboring antimicrobial resistance genes. Overlapping virulence-associated traits between avian-source E. coli and human extraintestinal pathogenic E. coli (ExPEC) suggest zoonotic potential and safety threat of poultry food products. We analyzed whole-genome sequencing (WGS) data of 46 mcr-1-positive E. coli strains isolated from retail raw meat purchased in the Czech Republic. The investigated strains were characterized by their phylogroup—B1 (43%), A (30%), D (11%), E (7%), F (4%), B2 (2%), C (2%), MLST type, and serotype. A total of 30 multilocus sequence types (STs), of which ST744 was the most common (11%), were identified, with O8 and O89 as the most prevalent serogroups. Using the VirulenceFinder tool, 3 to 26 virulence genes were detected in the examined strains and a total of 7 (15%) strains met the pathogenic criteria for ExPEC. Four strains were defined as UPEC (9%) and 18 (39%) E. coli strains could be classified as APEC. The WGS methods and available on-line tools for their evaluation enable a comprehensive approach to the diagnosis of virulent properties of E. coli strains and represent a suitable and comfortable platform for their detection. Our results show that poultry meat may serve as an important reservoir of strains carrying both virulence and antibiotic resistance genes for animal and human populations.

Variable Release of Lipoteichoic Acid From Staphylococcus aureus Bloodstream Isolates Relates to Distinct Clinical Phenotypes, Strain Background, and Antibiotic Exposure

BackgroundStaphylococcus aureus is a leading cause of bacterial bloodstream infections. The heterogeneity in patient outcomes in S. aureus bacteremia (SAB) can be attributed in part to strain characteristics, which may influence host response to infection. We specifically examined the relationship between lipoteichoic acid (LTA) release from S. aureus and disease phenotype, strain background, and antibiotic exposure.MethodsSeven strains of S. aureus causing different clinical phenotypes of bacteremia and two reference strains (LAC USA 300 and Mu3) were analyzed for LTA release at baseline and following exposure to antibiotics from different pharmacologic classes (vancomycin, ceftaroline, and tedizolid). LTA release was quantified by LTA-specific ELISA. Whole genome sequencing was performed on the clinical strains and analyzed using open-source bioinformatics tools.ResultsLipoteichoic acid release varied by 4-fold amongst the clinical strains and appeared to be related to duration of bacteremia, independent of MLST type. Low LTA releasing strains were isolated from patients who had prolonged duration of bacteremia and died. Antibiotic-mediated differences in LTA release appeared to be associated with MLST type, as ST8 strains released maximal LTA in response to tedizolid while other non-ST8 strains demonstrated high LTA release with vancomycin. Genetic variations related to the LTA biosynthesis pathway were detected in all non-ST8 strains, though ST8 strains showed no variations despite demonstrating differential LTA release.ConclusionOur findings provide the basis for future studies to evaluate the relationship between LTA release-mediated host immune response and clinical outcomes as well as the potential for antibiotic modulation of LTA release as a therapeutic strategy and deserve confirmation with larger number of strains with known clinical phenotypes.

Clinical and molecular characterization of Acinetobacter seifertii in Taiwan

Objectives Acinetobacter seifertii, a new member of the Acinetobacter baumannii group, has emerged as a cause of severe infections in humans. We investigated the clinical and molecular characteristics of A. seifertii. Patients and methods This retrospective study enrolled 80 adults with A. seifertii bloodstream infection (BSI) at four medical centres over an 8 year period. Species identification was confirmed by MALDI-TOF MS, rpoB sequencing and WGS. Molecular typing was performed by MLST. Clinical information, antimicrobial susceptibility and the mechanisms of carbapenem and colistin resistance were analysed. Transmissibility of the carbapenem-resistance determinants was examined by conjugation experiments. Results The main source of A. seifertii BSI was the respiratory tract (46.3%). The 28 day and in-hospital mortality rates of A. seifertii BSI were 18.8% and 30.0%, respectively. High APACHE II scores and immunosuppressant therapy were independent risk factors for 28 day mortality. The most common MLST type was ST553 (58.8%). Most A. seifertii isolates were susceptible to levofloxacin (86.2%), and only 37.5% were susceptible to colistin. Carbapenem resistance was observed in 16.3% of isolates, mostly caused by the plasmid-borne ISAba1-blaOXA-51-like genetic structure. A. seifertii could transfer various carbapenem-resistance determinants to A. baumannii, Acinetobacter nosocomialis and other A. seifertii isolates. Variations of pmrCAB and lpxCAD genes were not associated with colistin resistance of A. seifertii. Conclusions Levofloxacin and carbapenems, but not colistin, have the potential to be the drug of choice for A. seifertii infections. A. seifertii can transfer carbapenem-resistance determinants to other species of the A. baumannii group and warrants close monitoring.

Temporal Changes in Patient-Matched Staphylococcus epidermidis Isolates from Infections: towards Defining a ‘True’ Persistent Infection

Staphylococcus epidermidis is found naturally on the skin but is a common cause of persistent orthopaedic device-related infections (ODRIs). This study used a pan-genome and gene-by-gene approach to analyse the clonality of whole genome sequences (WGS) of 115 S. epidermidis isolates from 55 patients with persistent ODRIs. Analysis of the 522 gene core genome revealed that the isolates clustered into three clades, and MLST analysis showed that 83% of the isolates belonged to clonal complex 2 (CC2). Analysis also found 13 isolate pairs had different MLST types and less than 70% similarity within the genes; hence, these were defined as re-infection by a different S. epidermidis strain. Comparison of allelic diversity in the remaining 102 isolates (49 patients) revealed that 6 patients had microevolved infections (>7 allele differences), and only 37 patients (77 isolates) had a ‘true’ persistent infection. Analysis of the core genomes of isolate pairs from 37 patients found 110/841 genes had variations; mainly in metabolism associated genes. The accessory genome consisted of 2936 genes; with an average size of 1515 genes. To conclude, this study demonstrates the advantage of using WGS for identifying the accuracy of a persistent infection diagnosis. Hence, persistent infections can be defined as ‘true’ persistent infections if the core genome of paired isolates has ≤7 allele differences; microevolved persistent infection if the paired isolates have >7 allele differences but same MLST type; and polyclonal if they are the same species but a different MLST type.

Implementation and evaluation of different eradication strategies for Brachyspira hyodysenteriae

Background Brachyspira infections are causing major losses to the pig industry and lead to high antimicrobial use. Treatment of Brachyspira (B.) hyodysenteriae infections may be problematic due to the high level of antimicrobial resistance. The present study implemented and evaluated farm-specific eradication programmes for B. hyodysenteriae in 10 different infected pig farms in Belgium. Results Ten pig farms clinically infected with B. hyodysenteriae volunteered to implement a farm-specific eradication programme. The programme depended on the farm and management characteristics, antimicrobial susceptibility of the B. hyodysenteriae strain and the motivation of the farmer. Two farms practiced total depopulation, six farms partial depopulation and two farms antimicrobial medication without depopulation. In addition, all farms implemented biosecurity measures, and faeces samples were tested for the presence of B. hyodysenteriae at 6, 9 and 12 months after the start of the program. Single Brachyspira isolates from before and after the programme were typed using multilocus sequence typing (MLST). Eradication was successful in four farms. Two of them (farrow-to-finish and finishing herd) had applied total depopulation and respected a vacancy period of at least 3 weeks. A third farm (gilt farm) practised partial depopulation, the rooms remained empty for 28 days and changed the source of breeding gilts. The fourth farm practised partial depopulation, the stables remained empty for 3 weeks, and used antimicrobial medication. The eradication programme was not successful in six farms. Two of the latter farms only used medication without partial depopulation. Four farms practiced partial depopulation, one of them combined it with antimicrobial medication. The cleaning and disinfection procedures, rodent control, stand-empty period and/or other biosecurity measures in the six farms were not always implemented properly. In two of three farms, isolates belonging to the same MLST type were found before and after eradication. Conclusions Total depopulation or partial depopulation combined with implementing strict biosecurity measures allowed eradication of B. hyodysenteriae from clinically infected pig farms. Programmes based on antimicrobials without depopulation or partial depopulation without strictly adhering to all suggested biosecurity measures were not successful. Stockmanship and motivation of the farmer to permanently maintain high biosecurity standards are essential for success.

Marked Presence of Methicillin-Resistant Staphylococcus aureus in Wild Lagomorphs in Valencia, Spain

The appearance of methicillin-resistant strains of Staphylococcus aureus (MRSA) in several animal species (including rabbits) has set off alarms for their capacity to act as reservoirs for this bacterium. This is especially important in wild animals given its epidemiological implications. The objectives of this study were to identify and characterize S. aureus, specifically MRSA, strains in wild lagomorph high-density areas. Ten hares and 353 wild rabbits from 14 towns with a high rabbit density in the Valencian region (eastern Spanish coast) were sampled. Swabs from the nasal cavity, ears, perineum and lesions (when present) were taken for microbiological studies. The detection of different genes and antibiotic susceptibility studies were also carried out. Of all the animals, 41.3% were positive for S. aureus, of which 63.3% were MRSA. Ears were the anatomical location with more S. aureus and MRSA strains. The more frequently identified MLST type was ST1945 (97.1%, 136/140). The mecA gene was found only in one sample. The rest (n = 139) carried the mecC gene and were included in CC130, except one. Penicillin resistance was detected in 28 mec-negative isolates and, in one case, bacitracin resistance. mecA isolate presented resistance to enrofloxacin and tetracycline, and 10 mecC isolates also showed bacitracin resistance. No MRSA isolate was positive for genes chp, sea, tst and PVL. Two ST1945 isolates contained IEC type E (comprising genes scn and sak). mecA-isolate was positive for blaZ. Of the 28 MSSA strains showing resistance to penicillin, 22 carried the blaZ gene. These surprising results highlight the marked presence of MRSA strains in wild rabbits in high-density areas.

Characterization of the genotype and the phenotype of nontoxigenic strains of Corynebacterium diphtheriae subsp. lausannense isolated in Russian residents

In 2018, a few sequencing studies were published revealing the existence of two monophyletic clusters within the C. diphtheriae species, meaning that this species can be divided into two subspecies: C. diphtheriae subsp. diphtheriae and C. diphtheriae subsp. lausannense. The objective of our study was to describe the genotype and the phenotype of 2 nontoxigenic C. diphtheriae strains isolated in Russia in 2017–2018, which were classified by us as C. diphtheriae subsp. lausannense based on the aggregated data yielded by a variety of techniques, including microbiological and molecular genetic techniques, as well as a bioinformatic search for subspecies-specific genes in the publicly available genomes of C. diphtheriae. The isolated strains had morphological and biochemical characteristics of C. diphtheriae. The strains were assigned to the MLST type ST199 included in the clonal complex associated with subsp. lausannense. PCR revealed that both analyzed strains of C. diphtheriae subsp. lausannense carried the ptsI gene encoding phosphoenolpyruvate-protein phosphotransferase and did not carry the narG gene encoding the synthesis of nitrate reductase subunits, whereas the strains of C. diphtheriae subsp. diphtheriae had the narG gene and did not have ptsI. We experimentally proved the ability of lausannense strains to ferment N-acetylglucosamine. Our findings expand the knowledge of the biological diversity of C. diphtheriae and indicate the need for estimating the spread of these microorganisms in Russia, as well as their pathogenic potential.

The higher prevalence of extended spectrum beta-lactamases among Escherichia coli ST131 in Southeast Asia is driven by expansion of a single, locally prevalent subclone

The ST131 multilocus sequence type (MLST) of Escherichia coli is a globally successful pathogen whose dissemination is increasing rates of antibiotic resistance. Numerous global surveys have demonstrated the pervasiveness of this clone; in some regions ST131 accounts for up to 30% of all E. coli isolates. However, many regions are underrepresented in these published surveys, including Africa, South America, and Asia. We collected consecutive bloodstream E. coli isolates from three countries in Southeast Asia; ST131 was the most common MLST type. As in other studies, the C2/H30Rx clade accounted for the majority of ST131 strains. Clinical risk factors were similar to other reported studies. However, we found that nearly all of the C2 strains in this study were closely related, forming what we denote the SEA-C2 clone. The SEA-C2 clone is enriched for strains from Asia, particularly Southeast Asia and Singapore. The SEA-C2 clone accounts for all of the excess resistance and virulence of ST131 relative to non-ST131 E. coli. The SEA-C2 strains appear to be locally circulating and dominant in Southeast Asia, despite the intuition that high international connectivity and travel would enable frequent opportunities for other strains to establish themselves.