Fourier Transform Infrared Spectroscopy Is a New Option for Outbreak Investigation: a Retrospective Analysis of an Extended-Spectrum-Beta-Lactamase-Producing Klebsiella pneumoniae Outbreak in a Neonatal Intensive Care Unit.

The IR Biotyper is a new automated typing system based on Fourier-transform infrared (FT-IR) spectroscopy that gives results within 4 h. We aimed (i) to use the IR Biotyper to retrospectively analyze an outbreak of extended-spectrum beta-lactamase-producing Klebsiella pneumoniae (ESBL-KP) in a neonatal intensive care unit and to compare results to BOX-PCR and whole-genome sequencing (WGS) results as the gold standard and (ii) to assess how the cutoff values used to define clusters affect the discriminatory power of the IR Biotyper. The sample consisted of 18 isolates from 14 patients. Specimens were analyzed in the IR Biotyper using the default analysis settings, and spectra were analyzed using OPUS 7.5 software. The software contains a feature that automatically proposes a cutoff value to define clusters; the cutoff value defines up to which distance the spectra are considered to be in the same cluster. Based on FT-IR, the outbreak represented 1 dominant clone, 1 secondary clone, and several unrelated clones. FT-IR results, using the cutoff value generated by the accompanying software after 4 replicates, were concordant with WGS for all but 1 isolate. BOX-PCR was underdiscriminatory compared to the other two methods. Using the cutoff value generated after 12 replicates, the results of FT-IR and WGS were completely concordant. The IR Biotyper can achieve the same typeability and discriminatory power as genome-based methods. However, to attain this high performance requires either previous, strain-dependent knowledge about the optimal technical parameters to be used or validation by a second method.

Dissemination of the blaKPC gene by clonal spread and horizontal gene transfer: comparative study of incidence and molecular mechanisms.

OBJECTIVES: In addition to the global spread of the KPC-producing Klebsiella pneumoniae (KPC-KP) clonal complex (CC)-258 clone, the blaKPC gene may also spread by horizontal gene transfer (HGT), as suspected when more than one KPC-producing Enterobacteriaceae (KPC-Ent) species are isolated in a single patient. We aimed to characterize the incidence and molecular features of KPC-KP that were isolated alone (singular KPC-KP) versus KPC-KP that were isolated together with another KPC-Ent species (joint KPC-KP). METHODS: Isolates were collected from April 2011 to August 2012 at the Laniado Medical Center. Typing was done by CC-258 multiplex PCR and MLST. Plasmids were characterized by plasmid MLST (pMLST). The genetic environment of the blaKPC gene was studied by sequencing. RESULTS: During the 17 month period, there were 281 cases of singular KPC-KP and 8 cases of joint KPC-KP (P < 0.0001). Among the patients with joint KPC-KP, the additional KPC-Ent species were Escherichia coli (n = 6), Enterobacter aerogenes (n = 1), Enterobacter cloacae (n = 1) and Citrobacter freundii (n = 1). All singular KPC-KP isolates tested (n = 27) belonged to the CC-258 clone and carried the blaKPC-3 allele, located inside a Tn4401a transposon. In contrast, joint KPC-KP/KPC-Ent isolates belonged to different STs and all but one carried the blaKPC-2 allele. The blaKPC-2 gene was located inside ΔTn4401c transposons that were harboured by IncN/pMLST ST-15-type plasmids possessing high conjugation efficiency. CONCLUSIONS: This study highlights two dissemination modes of the blaKPC gene: clonal spread of the CC-258 clone and, far less commonly, HGT-related spread, mediated by ST-15 plasmids that shuttle between a variety of species and clones.

Emergence of VIM-producing Aeromonas caviae in Israeli hospitals.

OBJECTIVES: Resistance to carbapenems in Aeromonas species is rare and mediated mostly by the chromosomal cphA gene. Our aims were to describe the molecular characteristics of the first cases of VIM-producing Aeromonas caviae isolated from human samples. METHODS: Carbapenem-resistant Aeromonas (CRA) spp. were isolated from rectal surveillance cultures. Bacterial identification was done by dnaJ sequencing. Detection of metallo-carbapenemase and other ß-lactamase genes was done by PCR. Molecular typing was done by PFGE. The genetic environment of the blaVIM gene was determined by sequencing. RESULTS: Five CRA were isolated from surveillance cultures in 2010-13; four were from Shaare Zedek Medical Center and one was from Laniado Hospital. All five isolates were identified as A. caviae and comprised four different pulsotypes. MICs ranged from 0.5 to 8 mg/L for imipenem and from 0.25 to 8 mg/L for meropenem. All isolates were resistant to gentamicin, susceptible to amikacin and ciprofloxacin (except one), and were positive for carbapenemase production in the modified Hodge and Carba NP tests. The carbapenemase genes blaVIM-1 and blaVIM-35 were located inside a class I integron with two different sizes to its variable region. CONCLUSIONS: This is the first report of blaVIM in A. caviae from human samples and the first report of VIM-producing Gram-negative bacteria in Israel. This finding is alarming as this species may spread via water or sewage systems. Although infection due to Aeromonas spp. is rare, the presence of the gene on a mobile element is of concern due to the potential for dissemination to clinically important Gram-negative pathogens.

A swordless knight: epidemiology and molecular characteristics of the blaKPC-negative sequence type 258 Klebsiella pneumoniae clone.

In June 2010, a bla(KPC)-negative, ertapenem-resistant ST-258 Klebsiella pneumoniae strain was isolated from a patient in the Laniado Medical Center (LMC). Our aims were (i) to describe its molecular characteristics and resistance mechanisms and (ii) to assess whether the bla(KPC)-negative ST-258 K. pneumoniae clone spreads as efficiently as its KPC-producing isogenic strain. In a prospective study, surveillance of all ertapenem-resistant, carbapenemase-negative K. pneumoniae (ERCNKP) isolates was conducted from June 2010 to May 2011 at LMC (314 beds) and from July 2008 to December 2010 at the Tel Aviv Sourasky Medical Center (TASMC) (1,200 beds). Molecular typing was done by arbitrarily primed PCR, pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST). A total of 8 of 42 (19%) ERCNKP isolates in LMC and 1 of 32 (3.1%) in TASMC belonged to the ST-258 clone. These strains carried the bla(CTX-M-2) or the bla(CTX-M-25) extended-spectrum ß-lactamase (ESBL) gene. Sequencing of the ompK genes showed a frameshift mutation in the ompK35 gene. The fate of the bla(KPC)-carrying plasmid, pKpQIL, was determined by S1 analysis and by PCR of the Tn4401 transposon, repA, and the truncated bla(OXA-9). Plasmid analysis of the ERCNKP ST-258 isolates showed variability in plasmid composition and absence of the Tn4401 transposon and the pKpQIL plasmid. In addition, the ST-258 clone was identified in 35/35 (100%) of KPC-producing K. pneumoniae isolates but in none of 62 ertapenem-susceptible K. pneumoniae isolates collected in the two centers. Our results suggest that ERCNKP ST-258 evolved by loss of the bla(KPC)-carrying plasmid pKpQIL. ERCNKP ST-258 appears to have low epidemic potential.

High rate of bacterial respiratory tract co-infections upon admission amongst moderate to severe COVID-19 patients.

BACKGROUND: The role of bacterial and viral co-infection in the current COVID-19 pandemic remains elusive. The aim of this study was to describe the rates and features of co-infection on admission of COVID-19 patients, based on molecular and routine laboratory methods. METHODS: A retrospective study of COVID-19 and non-COVID-19 patients undergoing Biofire®, FilmArray® Pneumonia Panel, bioMérieux, and routine cultures during the first 3 days from admission, between June 2019 and March 2021. RESULTS: FilmArray tests were performed in 115 COVID-19 and in 61 non-COVID-19 patients. Most (>99%) COVID-19 patients had moderate-critical illness, 37% required mechanical ventilation. Sputa and endotracheal aspirates were the main samples analyzed. Positive FilmArray tests were found in 60% (70/116) of the tests amongst COVID-19 patients and 62.5% (40/64) amongst non-COVID-19 patients. All 70 cases were positive for bacterial targets, while one concomitant virus (Rhinovirus/Enterovirus) and one Legionella spp. were detected. The most common bacterial targets were Haemophilus influenzae (36%), Staphylococcus aureus (23%), Streptococcus pneumoniae (10%) and Enterobacter cloacae (10%). Correlation between FilmArray and cultures was found in 81% and 44% of negative and positive FA tests, respectively. Positive FilmArray results typically (81%) triggered the administration of antibiotic therapy and negative results resulted in antimicrobials to be withheld in 56% of cases and stopped in 8%. Bacterial cultures of COVID-19 patients were positive in 30/88 (34%) of cases. CONCLUSIONS: Bacterial co-infection is common amongst moderate-critical COVID-19 patients on admission while viral and atypical bacteria were exceedingly rare. Positive FilmArray results could trigger potentially unnecessary antibiotic treatment.KEY POINTWe found high rates of on-admission bacterial co-infection amongst hospitalized moderate to severe COVID-19 patients. Molecular tests (Biofire, FilmArray) and routine microbiological tests revealed 60% and 34% bacterial co-infection, respectively, while viral and fungal co-infections were rare.

Molecular Epidemiology of Methicillin-Resistant Staphylococcus aureus Clinical Isolates during 7.5 Years in One Regional Hospital in Israel.

Background: The clonal repertoire of community-associated Methicillin-resistant Staphylococcus aureus (CA-MRSA) strains appear to differ between hospitals and geographic locations. We aimed to study the molecular epidemiology of MRSA infections in our regional hospital in Israel. Methods: A retrospective analysis of MRSA isolates from hospitalized patients, which underwent spa typing between 2012 and 2019. Mainly, MRSA-bloodstream isolates were typed. Isolates were grouped into healthcare-associated (HcA) or community-associated (CA). HcA were further divided into hospital-related or long-term care facility- (LTCF-) related. Several representatives underwent SCCmec typing. Results: We analyzed 166 clinical MRSA isolates: 115 (70%) bloodstream, 42 (25%) wounds/abscesses, and 9 (5%) screening isolates. 145 (87%) were HcA, and 21 (13%) were CA. Common (72%) spa types were t002, t032, t008, t001, and t065. Eighty (55%) isolates were attributed to LTCFs and 65 isolates to our hospital, both showing similar spa types distribution. The most prevalent spa type among patients with HcA infection was t002 (50 isolates, 32%), followed by t032, t065, t578, t008, and t001. Most (88/115, 77%) bloodstream infections (BSIs) were HcA, typically occurring in the same facility in which the infection was acquired. In 27 cases (23%), the BSI developed in the community setting, and in half of these cases, a previous healthcare system exposure was evident. Conclusions: The MRSA clonal population in this longitudinal study was stable and consisted mainly of molecular lineages widespread in Europe. SCCmec-IV strains play a major role in causing MRSA infections in the healthcare settings, especially in LTCFs. Community-acquired MRSA BSIs without any previous healthcare exposure are still relatively rare.

Epidemiological, clinical and laboratory features of acute Q fever in a cohort of hospitalized patients in a regional hospital, Israel, 2012-2018.

INTRODUCTION: Acute Q fever is endemic in Israel, yet the clinical and laboratory picture is poorly defined. METHODS: A retrospective study reviewing the medical records of acute Q fever patients, conducted in a single hospital in the Sharon district, Israel. Serum samples from suspected cases were preliminary tested by a qualitative enzyme immunoassay (EIA). Confirmatory testing at the reference laboratory used an indirect immunofluorescence assay (IFA). Positive cases were defined as fever with at least one other symptom and accepted laboratory criteria such as a single-phase II immunoglobulin G (IgG) antibody titer ≥1:200. Cases not fulfilling these criteria and in which acute Q fever was excluded, served as a control group. RESULTS: Between January 2012 and May 2018, 484 patients tested positive. After confirmatory testing, 65 (13.4%) were positive for acute Q fever (with requisite clinical picture), 171 (35.3%) were definitely not infected, the remaining 248 were excluded because of past/chronic/undetermined infection. The average age was 58 years and 66% were males. Most resided in urban areas with rare animal exposure. Pneumonia was seen in 57% of cases and a combination with headache/hepatitis was highly suggestive of acute Q fever diagnosis. Syncope/presyncope, fall and arthritis were more common in acute Q fever cases. Laboratory indexes were similar to the control group, except for erythrocyte sedimentation rate (ESR) which was more common and higher in the study group. CONCLUSION: Acute Q fever in the Sharon district could be better diagnosed by using a syndromic approach in combination with improved rapid diagnostic testing.

High Rates of Bacterial Pulmonary Co-Infections and Superinfections Identified by Multiplex PCR among Critically Ill COVID-19 Patients.

BACKGROUND: The role of bacterial co-infection and superinfection among critically ill COVID-19 patients remains unclear. The aim of this study was to assess the rates and characteristics of pulmonary infections, and associated outcomes of ventilated patients in our facility. METHODS: This was a retrospective study of ventilated COVID-19 patients between March 2020 and March 2021 that underwent BioFire®, FilmArray® Pneumonia Panel, testing. Community-acquired pneumonia (CAP) was defined when identified during the first 72 h of hospitalization, and ventilator-associated pneumonia (VAP) when later. RESULTS: 148 FilmArray tests were obtained from 93 patients. With FilmArray, 17% of patients had CAP (16/93) and 68% had VAP (64/93). Patients with VAP were older than those with CAP or those with no infection (68.5 vs. 57-59 years), had longer length of stay and higher mortality (51% vs. 10%). The most commonly identified FilmArray target organisms were H. influenzae, S. pneumoniae, M. catarrhalis and E. cloacae for CAP and P. aeruginosa and S. aureus for VAP. FilmArray tests had high negative predictive values (99.6%) and lower positive predictive values (~60%). CONCLUSIONS: We found high rates of both CAP and VAP among the critically ill, caused by the typical and expected organisms for both conditions. VAP diagnosis was associated with poor patient outcomes.

Multidrug-resistant enterobacteriaceae in coastal water: an emerging threat.

BACKGROUND: The environmental role of carbapenemase-producing Enterobacteriaceae (CPE) acquisition and infection in human disease has been described but not thoroughly investigated. We aimed to assess the occurrence of CPE in nearshore aquatic bodies. METHODS: Enterobacteriaceae were cultured from coastal and estuary water near Netanya, Israel in June and July of 2018. Bacteria were identified by VITEK2® and their antimicrobial susceptibility was tested according to the CLSI guidelines. Enterobacteriaceae genomes were sequenced to elucidate their resistome and carbapenemase types. RESULTS: Among other clinically relevant bacteria, four CPE (three Enterobacter spp and one Escherichia coli isolate) were isolated from two river estuaries (Poleg and Alexander Rivers) and coastal water at a popular recreational beach (Beit Yanai). Molecular analysis and genome sequencing revealed the persistent presence of rare beta-lactamase resistance genes, including blaIMI-2 and a previously unknown blaIMI-20 allele, which were not found among the local epidemiological strains. Genome comparisons revealed the high identity of riverine and marine CPE that were cultivated one month apart. CONCLUSIONS: We show that CPE contamination was widespread in nearshore marine and riverine habitats. The high genome-level similarity of riverine and marine CPEs, isolated one month apart, hints at the common source of infection. We discuss the clinical implications of these findings and stress the urgent need to assess the role of the aquatic environment in CPE epidemiology.

Disseminated "Haemophilus quentini" infection in a patient with multiple myeloma - a case report and review of the literature.

We describe a case report of a 56-year-old male with undiagnosed multiple myeloma who had severe sepsis associated with pneumonia, meningitis, polyarthritis, and osteomyelitis related to invasive "Haemophilus quentini" infection. The genus was misidentified as H. influenzae by the common bacterial identification systems including newly introduced syndromic PCR-based methods. We review the epidemiological, clinical, and laboratory aspects of this rare, cryptic species of Haemophilus.

Screening asymptomatic households for Streptococcus pyogenes pharyngeal carriage as a part of in-hospital investigation of puerperal sepsis.

BACKGROUND: Invasive group A streptococcal (iGAS) infection in the peripartum setting is a rare but devastating disease occasionally occurring as a health care-associated infection (HAI). Current guidelines suggest enhanced surveillance and streptococcal isolate storage after a single case of iGAS, as well as a full epidemiological investigation that includes screening health care workers (HCWs) from several sites after 2 cases. Current guidelines do not recommend routine screening of household members of a patient with iGAS. METHODS: We conducted studies of 3 patients with iGAS puerperal sepsis and related epidemiologic and molecular investigations. RESULTS: Identical GAS emm gene types were found in pharyngeal cultures of 3 asymptomatic spouses of patients with iGAS puerperal sepsis. HCWs screened negative for GAS, and emm typing indicated that other iGAS cases from this hospital were sporadic and not related to the puerperal cases. CONCLUSIONS: The concurrent presence of the same emm type in a household member practically excludes the option of an inadvertent HAI or facility outbreak. Hence, we suggest that screening close family members for asymptomatic GAS carriage should be performed early as a part of infection prevention measures, as doing so would have significant utility in saving time and resources related to a full epidemiological inquiry.

Water faucets as a source of Pseudomonas aeruginosa infection and colonization in neonatal and adult intensive care unit patients.

We investigated the occurrence of Pseudomonas aeruginosa in our neonatal and adult intensive care units. Using enterobacterial repetitive intergenic consensus polymerase chain reaction, we showed spatial and temporal associations with clonal identity between patients' and adjacent faucets' clones. Both units' taps were highly colonized with P aeruginosa and with other waterborne bacteria. In the neonatal intensive care unit, strict use of sterile water for bathing neonates may have contributed to a reduction in clinical isolation of P aeruginosa postintervention.

A prospective survey of Pseudomonas aeruginosa colonization and infection in the intensive care unit.

BACKGROUND: Pseudomonas aeruginosa (PA) surveillance may improve empiric antimicrobial therapy, since colonizing strains frequently cause infections. This colonization may be 'endogenous' or 'exogenous', and the source determines infection control measures. We prospectively investigated the sources of PA, the clinical impact of PA colonization upon admission and the dynamics of colonization at different body sites throughout the intensive care unit stay. METHODS: Intensive care patients were screened on admission and weekly from the pharynx, endotracheal aspirate, rectum and urine. Molecular typing was performed using Enterobacterial Repetitive Intergenic Consensus Polymerase Chain reaction (ERIC-PCR). RESULTS: Between November 2014 and January 2015, 34 patients were included. Thirteen (38%) were colonized on admission, and were at a higher risk for PA-related clinical infection (Hazard Ratio = 14.6, p = 0.0002). Strains were often patient-specific, site-specific and site-persistent. Sixteen out of 17 (94%) clinical isolates were identical to strains found concurrently or previously on screening cultures from the same patient, and none were unique. Ventilator associated pneumonia-related strains were identical to endotracheal aspirates and pharynx screening (87-75% of cases). No clinical case was found among patients with repeated negative screening. CONCLUSION: PA origin in this non-outbreak setting was mainly 'endogenous' and PA-strains were generally patient- and site-specific, especially in the gastrointestinal tract. While prediction of ventilator associated pneumonia-related PA-strain by screening was fair, the negative predictive value of screening was very high.