Preliminary reading of antibiogram by microdilution for clinical isolates in urine culture.

PURPOSE: We evaluated a modification of automated antibiograms in urine cultures designed to facilitate the early interpretation of minimum inhibitory concentrations (MICs) and accelerate the targeted treatment of urinary tract infections (UTIs), METHODS: A prospective study was conducted of 309 isolates (219 Enterobacteriaceae, 75 Enterococcus spp., and 15 non-fermenting Gram-negative bacilli (NFGNB), and a retrospective study of 9 carbapenemase-producing clinical isolates from urine cultures. Colonies grown on conventional isolation plates were inoculated in MicroScan Walkaway system panels and incubated for 7 h, using a MicroScan AutoScan-4 plate reader for preliminary MIC determination by turbidimetry. Resulting antibiograms were compared with definitive antibiograms obtained after incubation for 17 h. RESULTS: Preliminary and definitive readings were concordant for 86.7% of Gram-positive cocci isolates (65/75), 61.6% of Enterobacteriaceae (135/219), and 53.3% of NFGNB. The agreement rate was greater than 90% for most antimicrobials against Gram-positive cocci (94.7% or more) and Enterobacteriaceae, (97.2% or more for 10 of 17 antibiotics) except with nitrofurantoin (89%). The agreement rate was 86.7% or more for most antibiotics against NFGNB apart from piperacillin/tazobactam, aztreonam, amikacin, and ciprofloxacin. Gram-negative bacilli showed the highest differences in MIC values between preliminary and definitive readings. CONCLUSIONS: A preliminary antibiogram reading may be useful in urine cultures to reduce the delay before targeted antibiotherapy, especially against Enterobacteriaceae and Gram-positive cocci, but not in cases of carbapenemase-producing NFGNB. Further local studies are warranted to evaluate the usefulness of this approach in relation to resistance rates.

Molecular epidemiology of carbapenem-resistant gram-negative bacilli in Ecuador.

INTRODUCTION: Carbapenem-resistant gram-negative bacilli are a worldwide concern because of high morbidity and mortality rates. Additionally, the increasing prevalence of these bacteria is dangerous. To investigate the extent of antimicrobial resistance and prioritize the utility of novel drugs, we evaluated the molecular characteristics and antimicrobial susceptibility profiles of carbapenem-resistant Enterobacterales, Pseudomonas aeruginosa and Acinetobacter baumannii in Ecuador in 2022. METHODS: Ninety-five clinical isolates of carbapenem non-susceptible gram-negative bacilli were collected from six hospitals in Ecuador. Carbapenem resistance was confirmed with meropenem disk diffusion assays following Clinical Laboratory Standard Institute guidelines. Carbapenemase production was tested using a modified carbapenemase inactivation method. Antimicrobial susceptibility was tested with a disk diffusion assay, the Vitek 2 System, and gradient diffusion strips. Broth microdilution assays were used to assess colistin susceptibility. All the isolates were screened for the blaKPC, blaNDM, blaOXA-48, blaVIM and blaIMP genes. In addition, A. baumannii isolates were screened for the blaOXA-23, blaOXA-58 and blaOXA-24/40 genes. RESULTS: Carbapenemase production was observed in 96.84% of the isolates. The blaKPC, blaNDM and blaOXA-48 genes were detected in Enterobacterales, with blaKPC being predominant. The blaVIM gene was detected in P. aeruginosa, and blaOXA-24/40 predominated in A. baumannii. Most of the isolates showed co-resistance to aminoglycosides, fluoroquinolones, and trimethoprim/sulfamethoxazole. Both ceftazidime/avibactam and meropenem/vaborbactam were active against carbapenem-resistant gram-negative bacilli that produce serin-carbapenemases. CONCLUSION: The epidemiology of carbapenem resistance in Ecuador is dominated by carbapenemase-producing K. pneumoniae harbouring blaKPC. Extensively drug resistant (XDR) P. aeruginosa and A. baumannii were identified, and their identification revealed the urgent need to implement strategies to reduce the dissemination of these strains.

Reliability of a Screening Method Using Antibiotic Disks to Detect Carbapenemases in Glucose-Nonfermenting Gram-Negative Microorganisms From Clinical Samples of a Regional Hospital in Southeastern Spain.

BACKGROUND: Infections by glucose-nonfermenting gram-negative bacilli (NFGNB) pose a major public health problem due to multiresistance to beta-lactam antibiotics, especially plasmid-borne carbapenemases. Their detection by microbiology laboratories is challenging, and there is a need for easy-to-use and reliable diagnostic techniques. Our objective was to evaluate an in-house screening method to presumptively detect carbapenemases in NFGNB in a simple and clinically useful manner. METHODS: The study included 175 NFGNB isolates from urinary, respiratory, and rectal samples. In a triple assay, isolates were incubated at 37°C for 24 h on three solid-culture media: MacConkey II Agar, 5% Sheep Blood Columbia Agar and Mueller Hinton II Agar; meropenem (MEM) and cefepime (FEP) disks were employed for screening. Studies were then performed on the inhibition halo diameter, scanning effects, and the appearance of mutant colonies, which were compared with those observed using the colorimetric Neo-Rapid CARB Kit and immunochromatography (NG5-Test Carba and K-Set for OXA-23). Receiver operating characteristic curves were constructed for these data. RESULTS: Carbapenemases were expressed by 79/175 (45.1%): 19 Pseudomonas aeruginosa and 60 Acinetobacter baumannii. Optimal inhibition halo diameter cutoffs to detect this resistance on 5% sheep blood agar were as follows: 6 mm (MEM) and 6.5 mm (FEP) for P. aeruginosa (in the absence of scanning effects and mutations) and 10.5 mm (MEM) and 16 mm (FEP) for A. baumannii (even in the presence of scanning effects). CONCLUSION: The combined utilization of MEM and FEP antibiotic disks in 5% sheep blood agar, measuring their inhibition haloes, offers an effective method to predict the presence of carbapenemases as resistance mechanism in P. aeruginosa and A. baumannii.

Genetic Characterization of Extensively Drug-Resistant Shigella sonnei Infections, Spain, 2021-2022.

In 2022, the United Kingdom reported an increase in drug resistance in Shigella sonnei isolates. We report 33 cases in Spain genetically related to the UK cases and 4 cases with similar antimicrobial resistance profiles infected with genetically distant strains. Our results suggest circulation of multiple genetic clusters of multidrug-resistant S. sonnei in Spain.

Identification of a Stable Chromosomal Tandem Multicopy of blaVIM-63, a New blaVIM-2 Carbapenemase.

This study characterizes a new genetic structure containing a multicopy of a blaVIM-2 variant with an A676C substitution, blaVIM-63. This gene was detected on the chromosome of two carbapenem-resistant clinical strains of Citrobacter freundii ST22 recovered from two patients, separated by a 6-month period, and previously in Pseudomonas aeruginosa ST2242 from the same hospital unit. Short-read sequencing was used to characterize the new variant in both species, and long-read sequencing was used to characterize the genome of C. freundii. On the P. aeruginosa chromosome, the blaVIM-63 gene was inserted between ISPsy 42-type sequences, flanked by an intl1 sequence, nearby aph(3')-VI, and sul1. On the C. freundii chromosome, the blaVIM-63 gene was inserted into a Tn6230-like transposon as a stable five-tandem-repeat multimer, flanked by the same intl1 as in P. aeruginosa. This structure was stable across subcultures and did not change in the presence of carbapenems. The blaVIM-63 gene was cloned into the pCR-Blunt plasmid to study antimicrobial susceptibility patterns and into pET29a for kinetic activity analysis. VIM-63 showed higher Km values than VIM-2 for ceftazidime and cefepime and higher kcat values for cefotaxime, ceftazidime, imipenem, and ertapenem, without differences in MIC values. This is the first study to describe this new variant, VIM-63, in two different species with a chromosomal location integrated into different mobile elements and the first to describe a stable multimer of a metallo-ß-lactamase. Despite the amino acid substitution, the susceptibility pattern of the new variant was similar to that of VIM-2. IMPORTANCE VIM group metallo-ß-lactamases are usually captured by IntI1 integrases. This work describes the detection for the first time of a novel, previously unknown variant of VIM-2, VIM-63. This carbapenemase has been found on the chromosome of two different species, Citrobacter freundii and Pseudomonas aeruginosa, from the same hospital. The adjacent genetic environment of the blaVIM-63 gene would indicate that the capture of this gene by IntI1 has occurred in two different genetic events in each of the species, and in one there has been a stable integration of tandem copies of this gene.

Colistin resistance screening by 3 µg/ml colistin agar in Carbapenemase-producing Enterobacterales.

BACKGROUND: In low- and middle-income countries, the use of colistin in therapeutic regimens is common, to treat infections produced for Carbapenemase-producing Enterobacterales (CPE) due to limited access to the recently discovered-approved antibiotics. Furthermore, the technical limitations to perform colistin susceptibility tests make it difficult to assess the suitability of this treatment for each patient, as well as to monitor the rates of resistance. In the present study, we describe the use of agar dilution using a unique colistin concentration of 3 µg/ml to discriminate isolates with colistin resistance in CPE obtained from clinical samples. METHODS: Clinical Laboratory Standards Institute (CLSI) colistin broth microdilution method and dilution agar with a colistin concentration of 3 µg/ml were performed in 168 isolates of CPE obtained from clinical samples in Guayaquil, Ecuador. Broth microdilution was considered our gold standard using CLSI breakpoints as reference (≤2 µg/ml intermediate and ≥4 µg/ml resistant). Categorical agreement was defined as obtaining a reading within the same category with both methodologies. RESULTS: Isolates obtained from respiratory samples were the most prevalent (26.19%; n = 44). Klebsiella pneumoniae was the predominant specie (94.04%; n = 158). KPC-like carbapenemase was present in all the isolates, and interestingly, colistin resistance was not mediated by MCR-1 production. Categorical agreement between both methods resulted in 97.02%. CONCLUSION: We propose the use of dilution agar with a colistin concentration of 3 µg/ml, as a valid method for screening colistin resistance in low- and middle-income countries to monitor resistance and to perform epidemiological studies.

Recent clinical relevance of mono-genital colonization/infection by Ureaplasma parvum.

Ureaplasma parvum is the most prevalent genital mycoplasma in women of childbearing age. There is debate around the relevance of its presence in male or female genitals for disease development and as a cofactor. The objective of this study was to determine the prevalence of colonization/infection by U. parvum and its possible relationship with reproductive tract infections. We retrospectively analyzed the presence of U. parvum in patients referred by specialist clinicians for suspicion of genitourinary tract infection. U. parvum was detected in 23.8% of samples, significantly more frequently in females (39.9%) than in males (6%). Among the males, U. parvum was found alone in 68.4% of episodes, with Ct < 30. Among the females, U. parvum was detected in 88.6% of cases, with Ct < 30, including 22 cases with premature rupture of membranes and 6 cases with threat of preterm labor. Co-infection was significantly more frequent in females (62.6%) than in males (31.6%). Given the high prevalence of U. parvum as sole isolate in males and females with genitourinary symptoms, it should be considered in the diagnosis and treatment of genital infections, although its pathogenic role in some diseases has not been fully elucidated.

Tracking KPC-3-producing ST-258 Klebsiella pneumoniae outbreak in a third-level hospital in Granada (Andalusia, Spain) by risk factors and molecular characteristics.

The objective of this study was to determine clinical-epidemiological characteristics of the patients and the genetic characteristics of carbapenemase KPC-3-producing Klebsiella pneumoniae isolates belonging to sequence type ST258. The eligible study population was all patients with isolates detected between October 2015 and March 2017. Clinical-epidemiological and microbiological data were gathered on risk factors associated with infection by this clone. Antimicrobial susceptibility was determined using MicroScan system and diffusion in agar. Genes encoding carbapenemases were detected using PCR and Sanger sequencing. The sequence type was assigned by MLST, and the genetic relationship among clinical isolates was determined by pulsed field electrophoresis and by analysis of the genetic environment. The study included 23 individuals with isolates of KPC-3/ST258; the mean age was 77 year, and mean stay pre-isolation was 32 days; 81% received empirical antimicrobial treatment. Isolates were only susceptible to gentamicin (CIM ≤ 2 mg/L), tigecycline (CIM ≤ 1 mg/L), and colistin (CIM ≤ 2 mg/L). The isolates belonged to ST258, with five pulse types or subgroups. All isolates showed amplification of KPC, which was identified as KPC-3 variant. Gene blaKPC-3 was flanked by insertion sequences Kpn6 and Kpn7 within Tn4401 transposon isoform a. We report, for the first time in Spain, an 18-month outbreak by KPC-3-producing ST258 K. pneumoniae. Its acquisition was associated with a history of antimicrobial therapy, with three treatment options, and with high mortality. The detection of different pulse types is attributable to different introductions of the clone in our setting, supporting the need for multi-resistant isolate surveillance studies.

Potential clinical use of azithromycin against gastroenteritis-causing pathogens other than Campylobacter.

The activity of azithromycin against enteritis-producing agents other than Campylobacter spp. was studied. The susceptibility to azithromycin, through gradient test, of 88 clinical isolates (51 Salmonella spp., 23 Aeromonas spp., 10 Shigella sonnei and 4 Yersinia enterocolitica) for one year was studied prospectively. The results were compared with the activity of ampicillin, trimethoprim-sulfamethoxazole and ciprofloxacin by microdilution. For azithromycin, the minimum inhibitory concentration (MIC) 50 and MIC90 were 4 and 12 mg/l, respectively. Six (6.8%) isolates were simultaneously resistant to ampicillin, trimethoprim- sulfamethoxazole and ciprofloxacin, and 3 (50%) of them presented a MIC >256 mg/l. Azithromycin may be a good empirical therapeutic option for the treatment of bacterial enteritis.

A propos of a new case of shigellosis by a non-imported multiresistant strain.

We present the case corresponding to a shigellosis produced by multiresistant S. flexneri in a patient with no recent history of tourism or travel to exotic countries. This case exposes the need to know the distribution of resistant strains, and their emergence not imported in our environment, in the face of choosing the most appropriate type of antibiotic, when appropriate.

[Epidemiological data description of pediatric patients with diarrhea by Aeromonas spp. and the antibiotic susceptibility of this agent]. / Descripción de datos clínico-epidemiológicos de pacientes pediátricos con diarrea por Aeromonas spp. y estudio de la sensibilidad antibiótica de dicho agente.

The aim of our study was to describe the epidemiological features of pediatric patients with diarrhea caused by Aeromonas spp. and to study the antibiotic susceptibility of this agent during a seven-year period. Aeromonas caviae was identified in 93 stool samples from 52.2% males and 85.6% patients younger than 36 months. The season with the lowest number of isolates was winter (14.4%). Co-infection with other diarrheagenic microorganisms was observed in 31.1% of the cases. The largest number of isolates was obtained from Emergency Department samples (45.6%); 43.3% of the patients presented with fever, 87.8% with diarrhea (43% of these cases were associated with pathological products) and 67.8% with vomiting, while 73.3% of the patients did not require hospital admission. Susceptibility higher than 87% was observed to trimethoprim-sulfamethoxazole, ciprofloxacin, cefotaxime and cefepime. All the patients overcame the infectious process and 63.3% of them did not receive any antibiotic treatment during the process. A. caviae was the isolated species associated with intestinal infection. Antibiotic treatment would be specifically indicated in cases selected for their clinical severity.

Gene pool transmission of multidrug resistance among Campylobacter from livestock, sewage and human disease.

The use of antimicrobials in human and veterinary medicine has coincided with a rise in antimicrobial resistance (AMR) in the food-borne pathogens Campylobacter jejuni and Campylobacter coli. Faecal contamination from the main reservoir hosts (livestock, especially poultry) is the principal route of human infection but little is known about the spread of AMR among source and sink populations. In particular, questions remain about how Campylobacter resistomes interact between species and hosts, and the potential role of sewage as a conduit for the spread of AMR. Here, we investigate the genomic variation associated with AMR in 168 C. jejuni and 92 C. coli strains isolated from humans, livestock and urban effluents in Spain. AMR was tested in vitro and isolate genomes were sequenced and screened for putative AMR genes and alleles. Genes associated with resistance to multiple drug classes were observed in both species and were commonly present in multidrug-resistant genomic islands (GIs), often located on plasmids or mobile elements. In many cases, these loci had alleles that were shared among C. jejuni and C. coli consistent with horizontal transfer. Our results suggest that specific antibiotic resistance genes have spread among Campylobacter isolated from humans, animals and the environment.

Emergent genital infection by Leptotrichia trevisanii.

We report the first case of an association between Leptotrichia trevisanii and an episode of pelvic inflammatory disease (PID) and the second case of the isolation of this infection in the cervical canal. A 45-yr-old woman was admitted to our emergency department with clinical and radiological signs and symptoms compatible with an episode of PID. She was hospitalized for intravenous antibiotic control and treatment and the subsequent surgical drainage of abscesses. Cultures were taken throughout the process, but only cultures from cervical canal exudate were positive, with the growth of L. trevisanii species. It appears important to carry out a complete microbiological screening, not limited to conventional agents, on adequate clinical samples to detect possible infectious agents that may be missed in these cases.

Alloscardovia omnicolens emerging presence in premature rupture of membranes.

Alloscardovia omnicolens is a recently-reported microorganism with unknown pathogenic implications. It has been isolated in various clinical localizations but not in the endocervix. We isolated A. omnicolens in an endocervical sample from a 31-yr-old patient with preterm premature rupture of membranes (PPROM) in week 33+3 of pregnancy. The main risk of PPROM is prematurity and the possibility of developing infectious chorioamnionitis, which can be lethal for the mother and newborn. This is the first report of an association between A. omnicolens and PPROM, although its pathogenic role has not yet been elucidated.

The first reported case of pelvic inflammatory disease caused by Actinobaculum massiliense.

We report the first case of pelvic inflammatory disease (PID) caused by Actinobaculum massiliense. A 53-year-old woman attended the emergency department with symptoms compatible with a PID episode, finally resolved by intramuscular antibiotic treatment. Actinobaculum sp. was isolated by culture, and A. massiliense was confirmed by matrix assisted laser desorption time-of-flight mass spectrometry and 16S rRNA gene sequencing. Only a few cases of A. massiliense infections have been reported, and the pathogenesis of infections by these bacteria is poorly understood. The introduction of new diagnostic methods into hospital routines will improve the detection of new and little-studied pathogens.

Susceptibility of clinical isolates of Campylobacter jejuni and Campylobacter coli to colistin.

Campylobacter spp. are one of the most frequent causes of bacterial diarrhea worldwide. Although severe diarrhea is not highly prevalent, the risk of a fatal outcome is increased when infection is caused by strains resistant to macrolides, fluoroquinolones, and/or tetracyclines. It is therefore necessary to test the susceptibility of these bacteria to other antibiotics such as colistin, which may serve as an alternative therapeutic option in these situations. The E-test was used to investigate the activity of erythromycin and colistin against 30 clinical isolates of Campylobacter spp. The MIC values obtained (range: 0.38-8 mg/liter) were sufficiently low, given the elevated concentrations that colistin sulfate can reach in the intestinal lumen, for this antibiotic to be considered useful to treat severe diarrhea caused by Campylobacter spp. resistant to first-line antibiotics.

Two Cases of Neisseria meningitidis Proctitis in HIV-Positive Men Who Have Sex with Men.

We report 2 cases from Spain of infectious proctitis caused by Neisseria meningitidis in HIV-positive men who have sex with men. Genetic characterization of the isolates showed that they are unusual strains not found in other more frequent meningococcal locations. This finding suggests an association between specific strains and anogenital tract colonization.

Analytical performance of the Alere™ i Influenza A&B assay for the rapid detection of influenza viruses.

The analytical performance of the new Alere™ i Influenza A&B kit (AL-Flu) assay, based on isothermal nucleic acids amplification, was evaluated and compared with an antigen detection method, SD Bioline Influenza Virus Antigen Test (SDB), and an automated real-time RT-PCR, Simplexa™ Flu A/B & VRS Direct assay (SPX), for detection of influenza viruses. An "in-house" RT-PCR was used as the reference method. Sensitivity of AL-Flu, SDB, and SPX was 71.7%, 34.8%, and 100%, respectively. Specificity was 100% for all techniques. The turnaround time was 13min for AL-Flu, 15min for SDB, and 75min for SPX. The Alere™ i Influenza A&B assay is an optimal point-of-care assay for influenza diagnosis in clinical emergency settings, and is more sensitive and specific than antigen detection methods.

The first description of the extended-spectrum beta-lactamase blaSHV-12 gene in a Salmonella monophasic Typhimurium strain isolated from acute gastroenteritis in a kidney transplant recipient in Southeast Spain.

Acute gastroenteritis (AGE) is a common disease within the population. Most cases are self-limited. However, intensive treatment is sometimes necessary to ensure patient integrity. This disease is characterized by vomiting and/or diarrhea with blood or mucus, discomfort, fever, and nonspecific abdominal pain. Commonly involved pathogens in the developed world include: viruses, bacteria, and parasites. In this paper we report a rare cause of AGE.