It is more convenient and practical to collect rectal swabs than stool specimens to study carriage of colon pathogens. In this study, we examined the ability to use rectal swabs rather than stool specimens to quantify Klebsiella pneumoniae carbapenemase (KPC)-producing carbapenem-resistant Enterobacteriaceae (CRE). We used a quantitative real-time PCR (qPCR) assay to determine the concentration of the bla KPC gene relative to the concentration of 16S rRNA genes and a quantitative culture-based method to quantify CRE relative to total aerobic bacteria. Our results demonstrated that rectal swabs are suitable for quantifying the concentration of KPC-producing CRE and that qPCR showed higher correlation between rectal swabs and stool specimens than the culture-based method. Klebsiella pneumoniae carbapenemase (KPC)-type enzymes are ␤-lactamases, capable of hydrolyzing all known ␤-lactam antibiotics (1). The spread of the bla KPC genes has led to the emergence of carbapenem-resistant Enterobacteriaceae (CRE), mostly K. pneumoniae, as important nosocomial pathogens causing outbreaks in various parts of the world (2). Infections by these pathogens are severe, with an estimated case fatality rate of 35%, and thus considered a clinical and public health threat (3).Active surveillance of high-risk patients has been advocated in areas where CRE are endemic or where there are ongoing outbreaks (4). Although stool samples are considered the "gold standard" specimen for studying gut bacteria in general (5, 6) and for detecting CRE in particular (7), hospital epidemiologists, clinical microbiologists, and researchers frequently use rectal swabs due to practical considerations, such as ease of collection, handling, and processing (8-11). Several studies compared the qualitative sensitivity of rectal swabs versus stool specimens for the recovery and detection of various pathogens, such as Campylobacter fetus subsp. jejuni, vancomycin-resistant Enterococcus faecium (VRE), and Lawsonia intracellularis, by culturing and/or molecular quantification methods (12)(13)(14). These studies had mixed results, with some studies reporting comparable test sensitivity with either sample type (12-14) and others reporting significantly lower sensitivity with rectal swabs (15, 16). For quantitative testing, rectal swabs are believed to be inadequate due to the high variation in the quantity of fecal material on each swab and the difficulties in determining this quantity (17)(18)(19)(20).As the human colon flora is a diverse ecosystem, which is populated by both anaerobic and aerobic microorganisms (21), development of quantitative methods, such as culture-and quantitative real-time PCR (qPCR)-based techniques is important for studying the bacterial composition of this complex ecosystem. The use of these methods can lead to a thorough knowledge about gastrointestinal community composition, the effects of antibiotics, and their roles in health and disease.Here, we suggest an alternate methodology for quantifying pathogens that would overcome the pr...

Objectives To analyse the epidemiology, the resistome and the virulome of ceftolozane/tazobactam-susceptible or -resistant Pseudomonas aeruginosa clinical isolates recovered from surveillance studies in Portugal (STEP, 2017–18) and Spain (SUPERIOR, 2016–17). Methods P. aeruginosa isolates were recovered from intra-abdominal, urinary tract and lower respiratory tract infections in ICU patients admitted to 11 Portuguese and 8 Spanish hospitals. MICs were determined (ISO-standard broth microdilution, EUCAST 2020 breakpoints). A subset of 28 ceftolozane/tazobactam-resistant P. aeruginosa isolates were analysed and compared with 28 ceftolozane/tazobactam-susceptible P. aeruginosa strains by WGS. Results Clonal complex (CC) 235 (27%) and CC175 (18%) were the most frequent, followed by CC244 (13%), CC348 (9%), CC253 (5%) and CC309 (5%). Inter-hospital clonal dissemination was observed, limited to a geographical region (CC235, CC244, CC348 and CC253 in Portugal and CC175 and CC309 in Spain). Carbapenemases were detected in 25 isolates (45%): GES-13 (13/25); VIM type (10/25) [VIM-2 (4/10), VIM-20 (3/10), VIM-1 (2/10) and VIM-36 (1/10)]; and KPC-3 (2/25). GES-13-CC235 (13/15) and VIM type-CC175 (5/10) associations were observed. Interestingly, KPC-3 and VIM-36 producers showed ceftolozane/tazobactam-susceptible phenotypes. However, ceftolozane/tazobactam resistance was significantly associated with GES-13 and VIM-type carbapenemase production. Six non-carbapenemase producers also displayed ceftolozane/tazobactam resistance, three of them showing known ceftolozane/tazobactam resistance-associated mutations in the PBP3 gene, ftsI (R504C and F533L). Overall, an extensive virulome was identified in all P. aeruginosa isolates, particularly in carbapenemase-producing strains. Conclusions GES-13-CC235 and VIM type-CC175 were the most frequent MDR/XDR P. aeruginosa clones causing infections in Portuguese and Spanish ICU patients, respectively. Ceftolozane/tazobactam resistance was mainly due to carbapenemase production, although mutations in PBP-encoding genes may additionally be involved.

CrpP enzymes have been recently described as a novel ciprofloxacin-resistance mechanism. We investigated by whole genome sequencing the presence of crpP-genes and other mechanisms involved in quinolone resistance in MDR/XDR-Pseudomonas aeruginosa isolates (n = 55) with both ceftolozane-tazobactam susceptible or resistant profiles recovered from intensive care unit patients during the STEP (Portugal) and SUPERIOR (Spain) surveillance studies. Ciprofloxacin resistance was associated with mutations in the gyrA and parC genes. Additionally, plasmid-mediated genes (qnrS2 and aac(6′)-Ib-cr) were eventually detected. Ten chromosomal crpP-like genes contained in related pathogenicity genomic islands and 6 different CrpP (CrpP1-CrpP6) proteins were found in 65% (36/55) of the isolates. Dissemination of CrpP variants was observed among non-related clones of both countries, including the CC175 (Spain) high-risk clone and CC348 (Portugal) clone. Interestingly, 5 of 6 variants (CrpP1-CrpP5) carried missense mutations in an amino acid position (Gly7) previously defined as essential conferring ciprofloxacin resistance, and decreased ciprofloxacin susceptibility was only associated with the novel CrpP6 protein. In our collection, ciprofloxacin resistance was mainly due to chromosomal mutations in the gyrA and parC genes. However, crpP genes carrying mutations essential for protein function (G7, I26) and associated with a restored ciprofloxacin susceptibility were predominant. Despite the presence of crpP genes is not always associated with ciprofloxacin resistance, the risk of emergence of novel CrpP variants with a higher ability to affect quinolones is increasing. Furthermore, the spread of crpP genes in highly mobilizable genomic islands among related and non-related P. aeruginosa clones alert the dispersion of MDR pathogens in hospital settings.

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The effect of a standard regimen of cimetidine on the gastric flora of 20 male volunteers was studied in a double-blind manner and compared with the effects of a standard antacid regimen. Postprandial microbial titers in gastric aspirates were significantly higher at 4, 8, and 16 weeks of therapy in subjects taking antacids and at 4 weeks in subjects taking cimetidine when compared with their pretreatment titers. Although not significant, there was a tendency for fasting microbial titers to be higher in subjects receiving cimetidine as compared with pretreatment titers. The higher titers were primarily related to increases in survival of mouth flora (viridans streptococci and Neisseria spp.); Enterobacteriaceae and other nitratereducing organisms were unusual isolates. There was no significant difference in the total titers or types of organisms isolated when subjects taking cimetidine were compared with those taking antacid. In 1939 Garrod demonstrated the bactericidal activity of hydrochloric acid and gastric juice (6). Others have subsequently reported that the type and numbers of microbial flora present in the stomach are affected by gastric pH (3, 8, 9). Drasar et al. (3) and Giannella et al. (7) showed that, unlike normal stomachs, which contain few bacteria, stomachs of patients with hypochlorhydria or achlorhydria maintain high bacterial counts. Bacterial overgrowth in the stomach increases the risk of wound sepsis after gastric surgery (14).