Molecular characterization of a multidrug-resistant/pandrug- resistant nosocomial polymicrobial infection with Klebsiella pneumoniae, Providencia rettgeri, and Acinetobacter baumannii from Rural Maharashtra, India

The emergence of resistance against commonly used antibiotics has become a serious global concern. The rapid development of antibiotic resistance exhibited by Enterobacteriaceae has caused an increasing concern regarding untreatable bacterial infections. Here, we isolated four pathogens from a geriatric female patient who was hospitalized for a month with ventilator-associated pneumonia (VAP) and fever. The organisms isolated from the tracheal aspirates and urine included Klebsiella pneumoniae, pandrug-resistant Providencia rettgeri, and Acinetobacter baumannii. Resistome analysis indicated that the bacterial isolates from the polymicrobial infection were multiple-drug resitnat and pandrug resistant clones. Molecular characterization revealed presence of blaTEM-1 in K. pneumonaie, P. rettgeri and A. baumannii. The blaTEM-1 and blaNDM-1 genes were present in P. rettgeri and A. baumannii, whereas the blaTEM-1, blaNDM-1 and blaOXA-23 traits were present in A. baumannii isolates. The patient has died due to the unavailability of effective antimicrobial treatment for this drug-resistant polymicrobial infection.


INTRODUCTION
Microorganisms that are primarily involved in antibiotic resistance are called the "ESKAPE" pathogens, and include Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, A. baumannii, Pseudomonas aeruginosa and Enterobacter species, capable of "escaping" from commonly used antibacterial treatments (Boucher et al., 2009). A. baumannii has emerged as a highly challenging pathogen due to its specific antibiotic resistance characteristics (Baucher et al,. 2009). Moreover, reports of extensively drug-resistant and pandrug-resistant K. pneumoniae (XDR-KP and PDR-KP) cases are increasing worldwide (Fiorellakrap et al., 2018). K. pneumoniae is the most clinically relevant Klebsiella species and is responsible for >70% of infections (Hansen et al., 1998). Antimicrobial resistance has become a global crisis because of escalating resistance coupled with diminished antibiotics in the developmental pipeline. A recent report estimates that by 2050, antimicrobial resistance-related mortality will be 10 000 000/ year (de Kraker et al., 2016).
The rapid emergence of carbapenem-resistant Enterobacteriaceae (CRE) worldwide has led to the concern that these infections may be soon untreatable. Management of infections caused by K. pneumoniae has been complicated by antimicrobial resistance, especially that against carbapenems. Whole genome sequence analyses of six extensively drug resistant (XDR) enteric pathogens isolated at New Delhi revealed multiple mobile genetic elements that were physically linked to antibiotic resistance traits. Thus, these elements seem to be responsible for disseminating drug resistance among organisms through underlying mechanisms of horizontal gene transfer and resistance to commonly used antibiotics (Kumar et al., 2017). Resistance to carbapenems in K. pneumonia involves multiple mechanisms, including production of carbapenemases, such as KPC, NDM, VIM, and OXA-48-like (Johann et al., 2015).
A 10-year study at Nashik, India (Odsbu et al., 2018, revealed a significantly higher proportion of non-susceptible and extended-spectrum β-lactamase (ESBL)-producing isolates from inpatients than those from outpatients for both, Escherichia coli and Klebsiella spp. A higher proportion of non-susceptible isolates indicates a great need to focus on the optimal use of antibiotics to reduce the development of antibiotic resistance.
Diverse risk factors associated with multidrug-resistance (MDR) in A. baumannii and other Enterobacteriaceae members suggest that a separate outbreak investigation should be performed in each hospital setting. Development of innovative control strategies is needed to limit the spread of these pathogens (Falagas & Kopterides, 2006).
In this study, we aimed to elucidate the mechanisms underlying drug resistance exhibited by prevalent pathogens responsible for unresponsiveness to the treatment administered to the patient. K. pneumoniae, P. rettgeri, and A. baumannii were isolated from the urine and tracheal aspirate of the patient on admission to the Somani Hospital, Barshi, Maharashtra, India.

CASE PRESENTATION
A 64-year-old female patient was hospitalized in Barshi with altered behavior, history of fall, and intracranial hemorrhage; the patient was put on a ventilator. Earlier, for 4 weeks, she received treatment at the Neurology Centre in Solapur, Maharashtra, and upon stabiliza- tion, she was moved to the Dr. Yogesh Somani Hospital, Barshi. During treatment, the patient developed a ventilator-associated pneumonia (VAP) and was administered piperacillin/tazobactam, meropenem, tigecycline, and colistin without a culture and susceptibility testing ( Fig. 1, first hospitalization).
On arrival at the Somani Hospital, the patient had a fever, was drowsy and arousable to the delayed premonition syndrome (DPS). She did not respond to verbal command, was on tracheostomy and discontinued from the ventilator use. She was treated with oral fluconazole and intravenous imipenem. Tigecycline was included in the treatment regimen without prior confirmation obtained by using an antibiogram or susceptibility testing. This therapy continued until the 16th day of hospitalization.
The patient was found to have bacteriuria and dead pus cells in the urine; therefore, urine culture and a susceptibility test was performed on the 32nd day of hospitalization; a mixed infection of Candida albicans and P. rettgeri was found. Based on susceptibility analysis, fluconazole, meropenem, and moxifloxacin was administered for the next 8 days. The fever continued and tigecycline was administered again for 7 days. On day 32, tracheal aspirate were tested for culture; K. pneumoniae and A. baumannii KSK0 were identified and isolated.
Based on the culture results and susceptibility analysis, treatment with meropenem and moxifloxacin was started. On day 47, she had high-grade fever, deteriorated CNS status, and was put on a ventilator support; the treatment was augmented and gentamicin was initiated in addition to meropenem and moxifloxacin.
On day 48, the culture and susceptibility analysis for the tracheal aspirate revealed A. baumannii KSK1, and gentamicin injections were administered along with the treatment on day 53, until the death of the patient on day 66. The cause of death was poly-microbial infection caused by resistant pathogens. Available antibiotics and treatment were insufficient.

MATERIALS AND METHODS
The isolates were collected during December 2017 at Barshi town in Maharashtra, India. Isolates were cultured on blood and MacConkey agars for purification. Well isolated, similar looking colonies were sub-cultured on trypticase soy agar and preserved in glycerol at -70°C for further analysis. Isolates were identified by the VITEK-2 (bioMérieux) system. Susceptibility testing. The four selected isolates: K. pneumoniae, A. baumannii KSK0, and A. baumannii KSK1 from tracheal aspirates, and P. rettgeri from urine, were susceptibility tested using the VITEK-2 (bioMérieux) system. The panel covered a broad range of antibiotics to estimate resistance and guide the antibiotic therapy. The isolates were susceptibility tested by also using a reference broth microdilution minimum inhibitory concentrations (MIC) determination method, as described by the CLSI (M07, A10). Briefly, a serial two fold dilutions of the antibacterial agents were made in cation-adjusted Muller-Hinton broth (BD, USA) in the presence or absence of a fixed inhibitor concentration. Bacterial suspensions of 0.5 McFarland turbid- Upon first admission, which was due to neurological complications, she was treated empirically for a month with Piperacillin/Tazobacta, Meropenem, Tigecyclin (a week) and Colistin. During the second hospitalization, culturing was performed for the tracheal fluid and one time culture was done from urine. Patient was found to have infected with yeast and three types of MDR, XDR and PDR bacterial pathogens.
ity equivalents were prepared in sterile 0.85% Saline (NaCl) and were appropriately diluted to obtain a final cell density of 2-8×10 5 CFU/mL in the antibiotic containing medium. The plates were incubated for 18 h at 37°C. MICs were recorded as the lowest antibiotic concentration showing no visible growth of an organism. Categorical interpretations for all comparator agents were those found in the CLSI breakpoint tables (M100, S26). Quality control was performed using Escherichia coli ATCC 25922. All quality control MIC results were within acceptable ranges published in CLSI documents. The antibiotic panel included ceftazidime in combination with avibactam to detect the presence of KPC, Ambler Class C, and OXA-48 enzymes while, meropenem with EDTA was included to determine the presence of a metallo-β-lactamase (MBL) enzyme.
Genotype Determination. All of the isolates were tested for the presence of bla CTX-M variants, bla TEM , bla SHV , bla KPC , bla CMY-2 , bla NDM-1 , and bla OXA 23/24 genes by a PCR assay using specific primers ( Table 1). The bacterial lysate was used as template DNA with a final reaction volume of 25 μl containing 10× buffer, 2.5 mM of dNTPs, 15 mM MgCl 2 , 100 pM of each oligonucleotide primer, 1 U of Taq polymerase and 2 μl of bacterial lysate. PCR was carried out in a thermal cycler using specific annealing temperatures as shown in Table 1. Amplified products were resolved in an agarose gel. The band size for the specific amplified genes was compared with the control samples in the same run.

RESULTS AND DISCUSSION
A female patient was admitted at the Dr. Yogesh Somani Hospital, Barshi, MS, India after a treatment course for neurological complications. Over a longer period of stay in the hospital, the patient developed serial episodes of nosocomial infections. Four isolates: K. pneumoniae and A. baumannii KSK0 from the initial, A. baumannii KSK1 from the terminal tracheal aspirate sample, and P. rettgeri from the second to last urine sample were isolated subsequently. Upon antibiotic susceptibility testing using VITEK-2, as well as broth microdilution, K. pneumoniae was found to be susceptible to the first line treatment agents, including the third and fourth generation cephalosporins, carbapenems, and aminoglycosides. The given treatment appeared effective amid no recovery of K. pneumoniae in subsequent tracheal aspirate cultures.
P. rettgeri was isolated from the patient's urine. This was attributed to the prolonged catheterization and nosocomial infection. Susceptibility testing showed that, P. rettgeri was resistant to all of the tested antibacterial agents except to meropenem-EDTA. Susceptibility to the meropenem-EDTA combination exhibited an MBL expression. Moreover, intrinsic resistance of P. rettgeri to a few antibiotic classes further limited the treatment options. Notably, both A. baumannii isolates from the tracheal aspirates were multidrug-resistant, except for their  (Table 2).

Resistotype and Genotyping studies
The VITEK-2 and broth microdilution MIC susceptibility results shown in Table 3 were validated by performing PCR amplifications for various β-lactamase traits. Results presented in Fig. 2 (a, b, c), and summarized in Table 4, show that the bla TEM-1 variant was present in all four clinical isolates, whereas bla NDM-1 was present only in P. rettgeri. Presence of bla NDM-1 explains the susceptibility of P. rettgeri to meropenem in combination with the metal ion chelating agent EDTA. Interestingly, bla OXA was found in A. baumannii KSK0 and A. baumannii KSK1, along with bla NDM-1 isolated at different stages.
All clinical isolates were genetically ESBL positive, however, A. baumannii KSK0 and A. baumannii KSK1 had three β-lactamases, indicating evolution of a complicated drug resistance mechanism.
Virulent and resistant K. pneumoniae strains are a significant cause of hospital-acquired infections. Studies performed in Iran reported a high prevalence of resistance against several antibiotics, and the simultaneous presence Table 2. Antibiotic susceptibility of pathogens isolated from the tracheal secretion and urine by VITEK-2

Klebsiella pneumoniae Providencia rettgeri
Acinetobacter baumannii KSK0  of certain virulence factors and MDR genes, contributing to a crucial public health issue (Ranjbar et al., 2019). Carbapenemases and ESBLs are responsible for pandrugresistance (PDR) in K. pneumoniae clinical samples in Maharashtra, India . In their study, ESBL resistance was observed in 310 (88.57%) isolates and carbapenemase in 181 (51.71%) isolates; these were the primary mechanisms underlying antibiotic resistance. A total of 29 (8.28%) K. pneumoniae PDR isolates and 52 (14.85%) isolates susceptible to colistin alone were found. They found extreme drug resistance in 135 (38.57%) of the K. pneumoniae isolates. The emergence and spread of MDR, ESBL producing carbapenem-resistant members of Enterobacteriaceae has become a worldwide health problem. A study in Bangkok, Thailand, revealed a unique prevalence of carbapenemase genes, where bla NDM-1 and bla OXA-232 were predominant (Laolerd et al., 2018).
Providencia species are intrinsically resistant to colistin and tigecycline, making the treatment of MDR Providencia spp. challenging. Carbapenem-resistant Providencia spp. have been reported (Abdallah & Balshi, 2018). An outbreak of carbapenem-resistant P. rettgeri, involving 4 patients admitted to intensive care and high-care units at a tertiary hospital was reported. Their clinical and demographic characteristics were studied; experiments revealed that all P. rettgeri strains were resistant to carbapenems -imipenem, ertapenem, and meropenem (Tshisevhe et al., 2016). Our results are in line with these referred studies that the P. rettgeri strain isolated from urine of our patient was a PDR strain.
The MDR A. baumannii has been recognized as clinically significant. Numerous reports relay the spread of A. baumannii in hospital settings, leading to nosocomial outbreaks with increased mortality. However, many Acinetobacter spp. can also cause nosocomial infections. A review focused on the role of Acinetobacter spp. as nosocomial pathogens along with their persistence, antimicrobial resistance patterns, and epidemiology has been recently published (Almasaudi, 2018).
Antimicrobial resistance among Acinetobacter spp is a global threat (Clark et al., 2016). A. baumannii is a major cause of healthcare-associated infections. MDR A. baumannii is a rapidly emerging pathogen, especially in the intensive care units, causing infections including bacteremia, pneumonia/VAP, meningitis, urinary tract infection, central venous catheter-related infection, and wound infection. An optimal treatment for A. baumannii nosocomial infections has not been established (Clark et al, 2016). However, the antibiotics that are usually effective against A. baumannii infections include carbapenems, polymyxins E and B, sulbactam, piperacillin/tazobactam, tigecycline, and aminoglycosides. Carbapenems (imipenem, meropenem, doripenem) are the mainstay of A. baumannii treatment; however, carbapenem-resistant Acinetobacter strains have been recently reported. These bacteria commonly present resistance to multiple antimicrobial agents, including carbapenems and polymyxins; hence, they are considered the paradigm of MDR or PDR bacteria. The XDR A. baumannii KSK0 and A. baumannii KSK1 isolates were difficult to treat as their MIC values were much higher than the prescribed doses for these antibiotics.
The indiscriminate and widespread antibiotic use causes rise of the resistant A. baumannii strains. A study performed in Chennai, India, reported the frequency of MDR (71.23%) and XDR (39.72%) for A. baumannii iso- MIC, minimum inhibitory concentration; ceftazidime with fixed clavulanic acid 4 mg/L, ceftazidime with fixed avibactam 4 mg/L, piperacillin with tazobactam 4 mg/L, EDTA, ethylenediaminetetraacetic acid at fixed 200 mg/L.